Compositions and Methods for Evaluating Viral Receptor/Co-Receptor Usage and Inhibitors of Virus Entry Using Recombinant Virus Assays

ABSTRACT

The present invention provides a method for identifying whether a compound inhibits entry of a virus into a cell which comprises: (a) obtaining nucleic acid encoding a viral envelope protein from a patient infected by the virus; (b) co-transfecting into a first cell (i) the nucleic acid of step (a), and (ii) a viral expression vector which lacks a nucleic acid encoding an envelope protein, and which comprises an indicator nucleic acid which produces a detectable signal, such that the first cell produces viral particles comprising the envelope protein encoded by the nucleic acid obtained from the patient; (c) contacting the viral particles produced in step (b) with a second cell in the presence of the compound, wherein the second cell expresses a cell surface receptor to which the virus binds; (d) measuring the amount of signal produced by the second cell in order to determine the infectivity of the viral particles; and (e) comparing the amount of signal measured in step (d) with the amount of signal produced in the absence of the compound, wherein a reduced amount of signal measured in the presence of the compound indicates that the compound inhibits entry of the virus into the second cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/926,837, filed Oct. 29, 2015, which is a continuation of U.S.application Ser. No. 12/635,539, filed Dec. 10, 2009, which is acontinuation of U.S. application Ser. No. 10/504,921 (abandoned), filedMar. 29, 2005, which is a U.S. National Stage under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/US2003/004373, filed Feb. 14,2003, which is a continuation of U.S. application Ser. No. 10/077,027,filed Feb. 15, 2002, (now U.S. Pat. No. 7,247,439), each of which areincorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Some aspects of the invention described in this application weresponsored by 1R21 DE019213-01A1 from the National Institute of Dentaland Craniofacial Research. Accordingly, the Federal Government hasrights in this application.

Throughout this application, various publications are referenced byauthor and date within the text. Full citations for these publicationsmay be found listed alphabetically at the end of the specificationimmediately preceding the claims. All patents, patent applications andpublications cited herein, whether supra or infra, are herebyincorporated by reference in their entirety. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“0956796_ST25.txt”, created on Feb. 3, 2016, and containing 11 KBytes.The material contained in this text file is incorporated by reference inits entirety for all purposes.

BACKGROUND OF THE INVENTION

Virus entry is an attractive new target for anti-viral treatment, andabout 10 drugs that are designed to block virus attachment or membranefusion are currently being evaluated in preclinical or clinical studies(Richman, 1998; PhRMA, 1999; Stephenson, 1999). Enveloped animal virusesattach to and enter the host cell via the interaction of viral proteinsin the virion membrane (envelope proteins) and cell surface proteins(virus receptors). Receptor recognition and binding are mediated by thesurface envelope protein.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide arapid, sensitive phenotypic assay to measure the susceptibility of avirus to inhibitors of viral entry.

A further object of the present invention is to provide a retroviralvector system that produces virus particles containing viral envelopeproteins derived from a variety of sources and the identification ofcell lines that express viral receptors and are permissive for viralreplication.

Another object of the present invention is to provide an expressionvector for viral envelope that is capable of accepting patient-derivedsegments encoding envelope genes.

Another object of this invention is to provide a bio-safe vector thatrepresents most of the HIV-1 viral genome, but carries a luciferasereporter gene in place of the envelope region.

A further object of the present invention is to the phenotypic assaywhich reduces the likelihood of forming recombinant infectious HIV-1, byproviding a viral expression vector that carries a deletion in atranscriptional regulatory region (the 3′ copy of U3) of the HD/4genome.

Another object of the present invention is to provide an assay capableof identifying and determining receptor/co-receptor tropism, whichquickly and accurately identifies patients that are infected withstrains of a tropic virus.

These and other objects may be achieved by the present invention by amethod for determining whether a virus has an altered susceptibility toa compound, said method comprising: (a) contacting a host cell with thecompound, wherein the host cell comprises a virus-derived nucleic acidand an indicator gene, the activity of the indicator gene is affected bythe activity of the virus-derived nucleic acid such that a change in theactivity of the virus-derived nucleic acid results in a change in theactivity of the indicator gene, and the compound directly or indirectlytargets the virus-derived nucleic acid or a protein it encodes, and (b)detecting the activity of the indicator gene, wherein a difference inthe activity of the indicator gene in the host cell contacted with thecompound relative to the activity in the absence of the compound,indicates that the virus has an altered susceptibility to the compound.

In one aspect of the invention, a first cell comprises a virus-derivednucleic acid encoding a viral protein and a viral expression vectorwhich lacks the nucleic acid encoding the viral protein and whichcomprises an indicator nucleic acid which produces a detectable signal,such that the first cell produces a viral particle comprising the viralprotein encoded by the virus-derived nucleic acid. The first cell can becontacted with a second cell, for example, a host cell, in the presenceof the compound, wherein the second cell expresses a cell surfacereceptor to which the viral particle binds. In one embodiment, thepresent invention provides a method for identifying whether a compoundinhibits entry of a virus into a cell which comprises: (a) obtainingnucleic acid encoding a viral envelope protein from a patient infectedby the virus; (b) co-transfecting into a first cell (i) the nucleic acidof step (a), and (ii) a viral expression vector which lacks a nucleicacid encoding an envelope protein, and which comprises an indicatornucleic acid which produces a detectable signal, such that the firstcell produces viral particles comprising the envelope protein encoded bythe nucleic acid obtained from the patient; (c) contacting the viralparticles produced in step (b) with a second cell in the presence of thecompound, wherein the second cell expresses a cell surface receptor towhich the virus binds; (d) measuring the amount of signal produced bythe second cell in order to determine the infectivity of the viralparticles; and (e) comparing the amount of signal measured in step (d)with the amount of signal produced in the absence of the compound,wherein a reduced amount of signal measured in the presence of thecompound indicates that the compound inhibits entry of the virus intothe second cell.

In another aspect, a first cell comprises a virus-derived nucleic acidencoding a viral protein and a viral expression vector which lacks thenucleic acid encoding the viral protein, such that the first cellproduces a viral particle comprising the viral protein encoded by thevirus-derived nucleic acid. The first cell can be contacted with asecond cell, for example, a host cell, in the presence of the compound,wherein the second cell expresses a cell surface receptor to which theviral particle binds. In addition, the second cell comprises anindicator nucleic acid which produces a detectable signal. In oneembodiment, the indicator nucleic acid is integrated into the nucleicacid of the second cell.

In one embodiment, the present invention provides a method for detectingwithin a patient infected by a virus the development of an antibodyresponse capable of blocking infection comprising: (a) contacting a hostcell with an antibody preparation from the patient, wherein the hostcell comprises a nucleic acid encoding a viral protein from the patientand an indicator nucleic acid which produces a detectable signal; (b)measuring the amount of the detectable signal produced by the host cell;and (c) comparing the amount of signal measured in step (b) with theamount of signal produced in the absence of the antibody preparation,wherein a reduced amount of signal measured in the presence of theantibody preparation indicates that the patient has developed anantibody response to the viral protein capable of blocking infection.

In another embodiment, the present invention provides a method fordetecting within a patient infected by a virus the development of anantibody response capable of blocking infection comprising: (a)transfecting into a first cell (i) a nucleic acid encoding a viralprotein from the patient, and (ii) a viral expression vector which lacksa nucleic acid encoding the viral protein, and which comprises anindicator nucleic acid which produces a detectable signal, such that thefirst cell produces viral particles comprising the viral protein encodedby the nucleic acid obtained from the patient; (b) contacting the viralparticles produced in step (a) with an antibody preparation from thepatient; (c) contacting the viral particles and antibody preparation ofstep (b) with a second cell, wherein the second cell expresses a cellsurface receptor to which the virus binds; (d) measuring the amount ofthe detectable signal produced by the second cell in order to determinethe infectivity of the viral particles; and (e) comparing the amount ofsignal measured in step (d) with the amount of signal produced in theabsence of the antibody preparation, wherein a reduced amount of signalmeasured in the presence of the antibody preparation indicates that thepatient has developed an antibody response to the viral protein capableof blocking infection.

In another embodiment, the present invention provides a method fordetecting within a patient infected by a virus the development of anantibody response capable of blocking infection comprising: (a)transfecting into a first cell (i) a nucleic acid encoding a viralprotein from the patient, and (ii) a viral expression vector which lacksa nucleic acid encoding the viral protein, such that the first cellproduces viral particles comprising the viral protein encoded by thenucleic acid obtained from the patient; (b) contacting the viralparticles produced in step (a) with an antibody preparation from thepatient; (c) contacting the viral particles and antibody preparation ofstep (b) with a second cell, wherein the second cell expresses a cellsurface receptor to which the virus binds and wherein the second cellcomprises an indicator nucleic acid which produces a detectable signal;(d) measuring the amount of the detectable signal produced by the secondcell in order to determine the infectivity of the viral particles; and(e) comparing the amount of signal measured in step (d) with the amountof signal produced in the absence of the antibody preparation, wherein areduced amount of signal measured in the presence of the antibodypreparation indicates that the patient has developed an antibodyresponse to the viral protein capable of blocking infection.

In one embodiment, the viral protein is an envelope protein. In anotherembodiment, the viral protein is a capsid protein.

In another embodiment, the present invention provides a method fordetecting within a patient infected by a virus the development of anantibody response capable of blocking infection comprising: (a)incubating a first cell comprising (i) a nucleic acid encoding a viralprotein from the patient, and (ii) a viral expression vector which lacksa nucleic acid encoding the viral protein, and which comprises anindicator nucleic acid which produces a detectable signal, such that thefirst cell produces viral particles comprising the viral protein encodedby the nucleic acid obtained from the patient; (b) contacting the viralparticles produced in step (a) with an antibody preparation from thepatient; (c) contacting the viral particles and antibody preparation ofstep (b) with a second cell, wherein the second cell expresses a cellsurface receptor to which the virus binds; (d) measuring the amount ofthe detectable signal produced by the second cell in order to determinethe infectivity of the viral particles; and (e) comparing the amount ofsignal measured in step (d) with the amount of signal produced in theabsence of the antibody preparation, wherein a reduced amount of signalmeasured in the presence of the antibody preparation indicates that thepatient has developed an antibody response to the viral protein capableof blocking infection. In one embodiment, the nucleic acid of (i) ispart of the viral expression vector of (ii). In another embodiment, thenucleic acid of (i) is integrated into the genome of the first cell. Inanother embodiment, the viral vector of (ii) is integrated into thegenome of the first cell. In another embodiment, the nucleic acid of (i)and the viral vector of (ii) are integrated into the genome of the firstcell. In one embodiment, the viral protein is a capsid protein. Inanother embodiment, the viral protein is an envelope protein.

BRIEF DESCRIPTION OF THE DRAWINGS

-   FIG. 1A: Structure of envelope expression and viral expression    vectors

The HIV envelope expression vector (pHIVenv) is modified to acceptenvelope sequences that have been amplified from patient plasma samples.The designations a/b and c/d, refer to restriction endonuclease sitespositioned at the 5′ and 3′ end of the HIV-1 envelope polyprotein(gp160). The HIV expression vector (pHIVlucΔU3) encodes all HIV proteinsexcept the envelope polyprotein. A portion of the envelope gene has beendeleted to accommodate a indicator gene cassette, in this case, “FireflyLuciferase” that is used to monitor the ability of the virus toreplicate in the presence or absence of anti-viral drugs. The 3′ U3region has been partially deleted to prevent transcription from the 5′LTR in infected cells. Virus produced in this system is limited to asingle round of replication.

-   FIG. 1B: Cell Based Entry Assay

Drug susceptibility, co-receptor tropism and virus neutralizationtesting are performed by co-transfecting a host cell with pHIVenv andpHIVlucΔU3. The host cell produces HIV particles that are pseudo-typedwith HIV envelope sequences derived from the test virus or patientsample. Virus particles are collected (˜48 h) after transfection and areused to infect target cells that express HIV receptors (e.g., CD4) andco-receptors (e.g., CXCR4, CCR5). After infection (˜72 h) the targetcells are lysed and luciferase activity is measured. HIV must completeone round of replication to successfully infect the target host cell andproduce luciferase activity. If the virus is unable to enter the targetcell, luciferase activity is diminished. This system can be used toevaluate susceptibility to entry inhibitors, receptor and co-receptortropism, and virus neutralization.

-   FIG. 2: HIV Envelope Expression Vectors

HIV envelope sequences are amplified from patient samples and insertedinto expression vectors using restriction endonuclease sites (5′ a/b and3′c/d). Envelope transcription is driven by the immediate early genepromoter of human cytomegalovirus (CMV). Envelope RNA is polyadenylatedusing an simian virus 40 (SV40) polyadenylation signal sequence (A+). Anintron located between the CMV promoter and the HIV envelope sequencesis designed to increase envelope mRNA levels in transfected cells. FLexpress full-length envelope proteins (gp120, gp41); ACT expressenvelope proteins (gp120, gp21) lacking the C-terminal cytoplasmic taildomain of gp41; +CT express envelope proteins (gp120, gp41) containing aconstant pre- defined gp41 cytoplasmic tail domain; gp120 express gp120proteins derived from the patient together with a constant pre-definedgp41; gp41 express a constant pre-defined gp120 together with gp41proteins derived from the patient.

-   FIG. 3A: Co-receptor Tropism Screening Assay

In this figure, the assay is performed using two cell lines. One cellline expresses CD4 and CCR5 (top six panels). The other cell lineexpresses CD4 and CXCR4 (bottom six panels). The assay is performed byinfecting cells with a large number of recombinant virus stocks derivedfrom cells transfected with pHIVenv and pHIVlucΔU3 vectors. The exampleshown represents the analysis of 96 viruses formatted in a 96 well plateinfections are performed in the absence of drug (no drug), or in thepresence of a drug that preferentially inhibits either R5 tropic (CCRinhibitor) or X4 tropic (CXCR4 inhibitor) viruses. Co-receptor tropismis assessed by comparing the amount of luciferase activity produced ineach cell type, both in the presence and absence of drug (see FIG. 3Bfor interpretation of assay results).

-   FIG. 3B: Determining co-receptor tropism

In this figure, the results of the assay are interpreted by comparingthe ability of each sample virus to infect (produce luciferase activity)in cells expressing CD4/CCR5 (R5 cells) or cells expressing CD4/CXCR4(X4 cells). The ability of a CCR5 or CXCR4 inhibitor to specificallyblock infection (inhibit luciferase activity) is also evaluated. X4tropic viruses (green panels)—infect X4 cells but not R5 cells.Infection of X4 cells is blocked by the CXCR4 inhibitor. R5 tropicviruses (blue panels)—infect R5 cells but not X4 cells. Infection of R5cells is blocked by the CCR5 inhibitor. Dual tropic or X4/R5 mixtures(yellow panels) infect X4 and R5 cells. Infection of R5 cells is blockedby the CCR5 inhibitor and infection of X4 cells is blocked by the CXCR4inhibitor. Non-viable viruses (red panels)—do not replicate in either X4or R5 cells.

-   FIG. 4A: Measuring Entry Inhibitor Susceptibility to Fusion    Inhibitor

In this figure, susceptibility to the fusion inhibitor T-20 isdemonstrated. Cells expressing CD4, CCR5 and CXCR4 were infected in theabsence of T-20 and over a wide range of T-20 concentrations (x-axislog10 scale). The percent inhibition of viral replication (y-axis) wasdetermined by comparing the amount of luciferase produced in infectedcells in the presence of T-20 to the amount of luciferase produced inthe absence of T-20. R5 tropic, X4 tropic and dual tropic viruses weretested. Drug susceptibility is quantified by determining theconcentration of T-20 required to inhibit 50% of viral replication(IC50, shown as vertical dashed lines). Viruses with lower IC50 valuesare more susceptible to T-20 than viruses with higher IC50 values.NL4-3: well-characterized X4 tropic strain JRCSF; well-characterized R5tropic strain 91US005.11: R5 tropic isolate obtained from the NIH AIDSResearch and Reference Reagent Program (ARRRP) 92HT593.1; Dual tropic(X4R5) isolate obtained from the NIH ARRRP.92HT599.24: X4 tropic isolateobtained from the NIH ARRRP.

-   FIG. 4B: Measuring Entry Inhibitor Susceptibility With Drug    Resistance Mutations

In this figure, reduced susceptibility to the fusion inhibitor T-20conferred by specific drug resistance mutations in the gp41 envelopeprotein is demonstrated. Cells expressing CD4, CCR5 and CXCR4 wereinfected in the absence of T-20 and over a wide range of T-20concentrations (x-axis log10 scale). The percent inhibition of viralreplication (y-axis) was determined by comparing the amount ofluciferase produced in infected cells in the presence of T-20 to theamount of luciferase produced in the absence of T-20. Isogenic virusescontaining one or two specific mutations in the gp41 transmembraneenvelope protein were tested (highlighted in red in the figure legend).Drug susceptibility is quantified by determining the concentration ofT-20 required to inhibit 50% of viral replication (IC50, shown asvertical dashed lines). Viruses with lower IC50 values are moresusceptible to T-20 than viruses with higher IC50 values.

No mutation (wildtype sequence): GIVSingle mutations: GIV, DIM, SIVDouble mutations: DIM, SIM, DTV

-   FIG. 5A: Measuring Entry Inhibitor Susceptibility to CCR5 Inhibitor

In this figure, susceptibility to a CCR5 inhibitor (merck compound) isdemonstrated. Cells expressing CD4 and CCR5 (R5 cells) were infected inthe absence of the CCR5 inhibitor and over a wide range of CCR5inhibitor concentrations (x-axis log10 scale). The percent inhibition ofviral replication (y-axis) was determined by comparing the amount ofluciferase produced in infected cells in the presence of CCR5 inhibitorto the amount of luciferase produced in the absence of CCR5 inhibitor.R5 tropic, X4 tropic and dual tropic viruses were tested. Drugsusceptibility is quantified by determining the concentration of CCR5inhibitor required to inhibit 50% of viral replication (IC50, shown asvertical dashed lines). Viruses with lower IC50 values are moresusceptible to the CCRF inhibitor than viruses with higher IC50 values.The X4 tropic virus did not infect the R5 cells. NL4-3:well-characterized X4 tropic strain.

JRCSF: well-characterized R5 tropic strain 92HT593.1: Dual tropic (X4R5)isolate obtained from the NIH ARRRP.

-   FIG. 5B: Measuring Entry Inhibitor Susceptibility to CXCR4 Inhibitor

In this figure, susceptibility to a CXCR4 inhibitor (AMD3100) isdemonstrated. Cells expressing CD4 and CXCR4 (X4 cells) were infected inthe absence of the CXCR4 inhibitor and over a wide range of CXCR4inhibitor concentrations (x-axis log10 scale). The percent inhibition ofviral replication (y-axis) was determined by comparing the amount ofluciferase produced in infected cells in the presence of CXCR4 inhibitorto the amount of luciferase produced in the absence of CXCR4 inhibitor.R5 tropic, X4 tropic and dual tropic viruses were tested. Drugsusceptibility is quantified by determining the concentration of CXCR4inhibitor required to inhibit 50% of viral replication (IC50, shown asvertical dashed lines). Viruses with lower IC50 values are moresusceptible to the CCR5 inhibitor than viruses with higher IC50 values.The R5 tropic virus did not infect the X4 cells.

NL4-3: well-characterized X4 tropic strainJRCSF: well-characterized R5 tropic strain92HT593.1: Dual tropic (X4R5) isolate obtained from the NIH ARRRP.

-   FIG. 6: Envelope Sequence Amplification

This figure demonstrates that the amplicons corresponding to the fulllength envelope sequence or cytoplasmic-tail deleted envelope sequenceare generated. The lane numbers correspond to the co-receptor tropismshown next to each number on the right of the gels.

-   FIG. 7: Target Cell Receptor and Co-Receptor Expression

Scatter plots are shown which indicate the results from FACS(fluorescence activated cell sorting) assays using antibodies againsteither CCR5 or CXCR4 (shown on Y axis). The cell lines express theco-receptors listed below the plots and the CD4 fluorescence is shownalong the X-axis. The anti-CXCR4 antibody binds most strongly with thecells which express the corresponding co-receptor, CXCR-4.

-   FIG. 8: Inhibition by Co-Receptor Antagonists

X4 and R5 inhibition is shown following administration of co-receptorantagonists.

-   FIG. 9: Fusion Inhibitor Peptides

CXCR4 inhibitor map and amino acid sequence is shown for a peptide whichis an inhibitor of fusion between a viral membrane and a cell membrane.

-   FIG. 10: Neutralization of Viral Infectivity

Viruses were incubated with serial 5-fold dilutions of antibody (plasma)and used to infect U-87/CD4/CCR5/CXCR4 target cells. Serial virussamples (columns) were tested against serial antibody samples (rows)using a matrix format. Neutralization values represent the plasma(antibody) dilution required to inhibit virus infectivity by 50% (IC50).The larger the number, the larger the dilution, reflecting higherantibody neutralization titers. Sample collection dates are representedas mm/dd/yy. The neutralizing activity of each plasma sample was alsotested against two reference viruses: NL4-3 (X4 laboratory strain), JRCSF (R5 primary isolate).

The present invention in its particular features can become moreapparent from the following detailed description considered withreference to the accompanying figures and examples. The followingdescription discusses the means and methods to carry out the presentinvention pertaining to a phenotypic assay relating to identifying andevaluating inhibitors of viral entry, including for example, and not asa limitation to the present invention, HIV-1 and inhibitors to HIV-1viral entry.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for determining whether a virus has analtered susceptibility to a compound, said method comprising: (a)contacting a host cell with the compound, wherein the host cellcomprises a virus-derived nucleic acid and an indicator gene, theactivity of the indicator gene is affected by the activity of thevirus-derived nucleic acid such that a change in the activity of thevirus-derived nucleic acid results in a change in the activity of theindicator gene, and the compound directly or indirectly targets thevirus-derived nucleic acid or a protein it encodes, and (b) detectingthe activity of the indicator gene, wherein a difference in the activityof the indicator gene in the host cell contacted with the compoundrelative to the activity in the absence of the compound, indicates thatthe virus has an altered susceptibility to the compound.

In one embodiment, the invention provides a method for identifyingwhether a compound inhibits entry of a virus into a cell whichcomprises: (a) obtaining nucleic acid encoding a viral envelope proteinfrom a patient infected by the virus; (b) co-transfecting into a firstcell (i) the nucleic acid of step (a), and (ii) a viral expressionvector which lacks a nucleic acid encoding an envelope protein, andwhich comprises an indicator nucleic acid which produces a detectablesignal, such that the first cell produces viral particles comprising theenvelope protein encoded by the nucleic acid obtained from the patient;(c) contacting the viral particles produced in step (b) with a secondcell in the presence of the compound, wherein the second cell expressesa cell surface receptor to which the virus binds; (d) measuring theamount of signal produced by the second cell in order to determine theinfectivity of the viral particles; and (e) comparing the amount ofsignal measured in step (d) with the amount of signal produced in theabsence of the compound, wherein a reduced amount of signal measured inthe presence of the compound indicates that the compound inhibits entryof the virus into the second cell.

In another embodiment, the invention provides a method for identifyingwhether a compound inhibits entry of a virus into a cell whichcomprises: (a) obtaining nucleic acid encoding a viral envelope proteinfrom a patient infected by the virus; (b) co-transfecting into a firstcell (i) the nucleic acid of step (a), and (ii) a viral expressionvector which lacks a nucleic acid encoding an envelope protein, suchthat the first cell produces viral particles comprising the envelopeprotein encoded by the nucleic acid obtained from the patient; (c)contacting the viral particles produced in step (b) with a second cellin the presence of the compound, wherein the second cell expresses acell surface receptor to which the virus and wherein the second cellcomprises an indicator nucleic acid which produces a detectable signalbinds; (d) measuring the amount of signal produced by the second cell inorder to determine the infectivity of the viral particles; and (e)comparing the amount of signal measured in step (d) with the amount ofsignal produced in the absence of the compound, wherein a reduced amountof signal measured in the presence of the compound indicates that thecompound inhibits entry of the virus into the second cell.

In another embodiment of this invention, the indicator nucleic acidcomprises an indicator gene. In another embodiment of this invention,the indicator gene is a luciferase gene.

In one embodiment of this invention, the cell surface receptor is CD4.In one embodiment of this invention, the cell surface receptor is achemokine receptor. In one embodiment of this invention, the cellsurface receptor is CXCR4 or CCR5.

In one embodiment of this invention, the patient is infected with theHIV-1 virus, a hepatitis virus (such as the HCV or HBV virus), or anyother virus. In one embodiment of this invention, the nucleic acid ofstep (a) comprises DNA encoding gp120 and gp41. In one embodiment ofthis invention, the viral expression vector comprises HIV nucleic acid.In one embodiment of this invention, the viral expression vectorcomprises an HIV gag-pol gene. In one embodiment of this invention, theviral expression vector comprises DNA encoding vif, vpr, tat, rev, vpu,and nef.

In one embodiment of this invention, the first cell is a mammalian cell.In one embodiment of this invention, the mammalian cell is a human cell.In one embodiment of this invention, the human cell is a human embryonickidney cell. In one embodiment of this invention, the human embryonickidney cell is a 293 cell.

In one embodiment of this invention, the second cell is a human T cell.In one embodiment of this invention, the second cell is a human T cellleukemia cell line. In one embodiment of this invention, the second cellis a peripheral blood mononuclear cell. In one embodiment of thisinvention, the second cell is an astroglioma cell. In one embodiment ofthis invention, the astrogliorna cell is a U87 cell. In one embodimentof this invention, the second cell is a human osteosarcoma cell. In oneembodiment of this invention, the human osteosarcoma cell is an HT4cell.

In one embodiment of this invention, the compound binds to the cellsurface receptor. In one embodiment of this invention, the compound is aligand of the cell surface receptor. In one embodiment of thisinvention, the compound comprises an antibody. In one embodiment of thisinvention, the compound inhibits membrane fusion. In one embodiment ofthis invention, the compound is a peptide, a peptidomimetic, an organicmolecule, or a synthetic compound. In one embodiment of this invention,the compound binds the viral envelope protein.

This invention provides for a method for making a composition whichcomprises admixing the compound identified by the screening method(method for identifying a compound) described herein with a carrier. Inone embodiment of this invention, the carrier is saline, polyethyleneglycol, a buffer solution, a starch, or an organic solvent.

The present invention provides for a method for identifying a cellsurface receptor which is bound by a virus upon infection of a cell bythe virus which comprises: (a) obtaining viral particles which comprise(i) a viral nucleic acid and (ii) an indicator nucleic acid whichproduces a detectable signal; (b) contacting a cell which expresses acell surface receptor with the viral particles from step (a); and (c)measuring the amount of detectable signal produced within the cell,wherein production of the signal indicates the cell surface receptorexpressed by the cell is bound by the virus, thereby identifying thecell surface receptor as being bound by the virus upon infection of thecell.

The present invention also provides for a method for identifying whetheran antibody inhibits entry of a virus into a cell which comprises: (a)obtaining nucleic acid encoding a viral envelope protein from a patientinfected by the virus; (b) co-transfecting into a first cell (i) thenucleic acid of step (a), and (ii) a viral expression vector which lacksa nucleic acid encoding an envelope protein, and which comprises anindicator nucleic acid which produces a detectable signal, such that thefirst cell produces viral particles comprising the envelope proteinencoded by the nucleic acid obtained from the patient; (c) contactingthe viral particles produced in step (b) with a second cell in thepresence of the antibody, wherein the second cell expresses a cellsurface receptor to which the virus binds; (d) measuring the amount ofsignal produced by the second cell in order to determine the infectivityof the viral particles; and (e) comparing the amount of signal measuredin step (d) with the amount of signal produced in the absence of thecompound, wherein a reduced amount of signal measured in the presence ofthe antibody indicates that the antibody inhibits entry of the virusinto the second cell.

The present invention provides for a method for determiningsusceptibility of a virus to a compound which inhibits viral cell entrywhich comprises: (a) obtaining nucleic acid encoding a viral envelopeprotein from a patient infected by the virus; (b) co-transfecting into afirst cell (i) the nucleic acid of step (a), and (ii) a viral expressionvector which lacks a nucleic acid encoding an envelope protein, andwhich comprises an indicator nucleic acid which produces a detectablesignal, such that the first cell produces viral particles comprising theenvelope protein encoded by the nucleic acid obtained from the patient;(c) contacting the viral particles produced in step (b) with a secondcell in the presence of the compound, wherein the second cell expressesa cell surface receptor to which the virus binds; (d) measuring theamount of signal produced by the second cell in order to determine theinfectivity of the viral particles; and (e) comparing the amount ofsignal measured in step (d) with the amount of signal produced in theabsence of the compound, wherein a reduced amount of signal measured inthe presence of the compound indicates that the virus is susceptible tothe compound.

The present invention provides a method for determining whether a virushas an increased resistance to a compound which inhibits viral entryinto a cell compared to a reference virus, said method comprising: (a)determining susceptibility of a virus to a compound, for example,according to the method described above, at a first time; (b)determining susceptibility of the virus to the compound at a latersecond time; and (c) comparing the susceptibilities determined in steps(a) and (b), wherein a decrease in susceptibility at the later secondtime indicates an increased resistance of the virus to the compound.

The present invention provides for a method for identifying a mutationin a virus that confers resistance to a compound that inhibits viralentry into a cell which comprises: (a) determining the nucleic acidsequence or the amino acid sequence of the virus prior to any treatmentof the virus with the compound; (b) obtaining a virus resistant to thecompound; (e) determining the nucleic acid sequence or the amino acidsequence of the resistant virus from step (b); and (d) comparing thenucleic acid sequence or the amino acid sequences of steps (a) and (c),respectively, so as to identify the mutation in the virus that confersresistance to the compound.

In one embodiment of this invention, the virus obtained in step (b) isthe virus of step (a) grown in the presence of the compound untilresistance is developed.

In one embodiment of this invention, the virus obtained in step (b) isisolated from a patient which has been undergoing treatment with thecompound.

In a preferred embodiment, this invention provides a means and methodfor accurately and reproducibly measuring the susceptibility of HIV-1 tovirus entry inhibitors.

In another preferred embodiment, this invention also provides a meansand method for accurately and reproducibly measuring HIV-1 co-receptortropism.

In a preferred embodiment, this invention provides a means and methodfor accurately and reproducibly measuring antibody mediatedneutralization of HIV-1.

In a preferred embodiment, this invention further provides a means andmethod for discovering, optimizing and characterizing novel or new drugsthat target various defined and as yet undefined steps in the virusattachment and entry process.

In a preferred embodiment, this invention further provides a means andmethod for discovering, optimizing and characterizing HIV-1 vaccines(either preventative or therapeutic) that target various defined and asyet undefined steps in the virus attachment and entry process.

In a preferred embodiment, this invention provides a means and methodfor identifying amino acid substitutions/mutations in HIV-1 envelopeproteins (gp41TM and gp120SU) that alter susceptibility to inhibitors ofvirus entry.

In a preferred embodiment, this invention provides a means and methodfor quantifying the affect that specific mutations in HIV-1 envelopehave on virus entry inhibitor susceptibility.

In a preferred embodiment, this invention further provides a means andmethod for determining HIV-1 envelope amino acid substitutions/mutationsthat are frequently observed, either alone or in combination, in virusesthat exhibit altered susceptibility to virus entry inhibitors.

In a preferred embodiment, this invention provides a means and methodfor identifying amino acid substitutions/mutations in HIV-1 envelopeproteins (gp41TM and gp120SU) that alter receptor or co-receptortropism.

In a preferred embodiment, this invention provides a means and methodfor quantifying the affect that specific mutations in HIV-1 envelopehave receptor or co-receptor tropism.

In a preferred embodiment, this invention further provides a means andmethod for identifying HIV-1 envelope amino acid substitutions/mutationsthat are frequently observed, either alone or in combination, in virusesthat exhibit CXCR4 or CCR5 co-receptor tropism.

In a preferred embodiment, this invention provides a means and methodfor identifying amino acid substitutions/mutations in HIV-1 envelopeproteins (gp41TM and gp120SU) that alter antibody mediatedneutralization.

In a preferred embodiment, this invention provides a means and methodfor quantifying the affect that specific mutations in HIV-1 envelopehave on antibody mediated neutralization.

In a preferred embodiment, this invention further provides a means andmethod for identifying HIV-1 envelope amino acid substitutions/mutationsthat are frequently observed, either alone or in combination, in virusesthat exhibit antibody medicated virus neutralization.

In a preferred embodiment, this invention further provides a means andmethod to identify antibodies that are frequently observed in patientsamples viruses that are capable of neutralizing HIV-1.

In a preferred embodiment, this invention further provides a means andmethod for identification of viruses that require CD4 binding forinfection.

In a preferred embodiment, this invention further provides a means andmethod for the identification of viruses that do not require CD4 bindingfor infection.

In a preferred embodiment, this invention also provides a means andmethod for identifying the incidence of patient samples that exhibit CD4independent infection.

In a preferred embodiment, this invention further provides a means andmethod for identification of viruses that require CD8 binding forinfection.

In a preferred embodiment, this invention also provides a means andmethod for identifying the incidence of patient viruses that exhibit CD8dependent infection.

In a preferred embodiment, this invention further provides the means andmethod for the identification of viruses that require the CXCR4chemokine receptor binding, the CCR5 chemokine receptor binding, oreither CXCR4 or CCR5 binding (dual tropic) for infection.

In a preferred embodiment, this invention further provides a means andmethod for identifying the incidence of viruses that require the CXCR4chemokine receptor binding, the CCR5 chemokine receptor binding, oreither CXCR4 or CCR5 binding (dual tropic) for infection.

In a preferred embodiment, this invention further provides a means andmethod for identifying HIV-1 envelope amino acid substitutions/mutationsthat are frequently observed, either alone or in combination, in virusesthat exhibit (a) altered susceptibility to virus entry inhibitors, (b)CXCR4 or CCR5 co-receptor tropism, and (c) antibody medicated virusneutralization.

In a preferred embodiment, this invention provides a means and methodfor using virus entry inhibitor susceptibility to guide the treatment ofHIV-1.

In a preferred embodiment, this invention further provides a means andmethod for using virus entry inhibitor susceptibility to guide thetreatment of patients failing antiretroviral drug treatment.

In a preferred embodiment, this invention further provides the means andmethods for using virus entry inhibitor susceptibility to guide thetreatment of patients newly infected with HIV-1.

In a preferred embodiment, this invention provides a means and methodfor using HIV-1 co-receptor tropism to guide the treatment of HIV-1 orto guide the treatment of patients failing antiretroviral drugtreatment.

In a preferred embodiment, this invention further provides the means andmethod for using HIV-1 co-receptor tropism to guide the treatment ofpatients newly infected with HIV-1.

In a preferred embodiment, this invention further provides a means andmethod for measuring antibody mediated neutralization of HIV-1 tomonitor the initial protective antibody response following vaccination.

In a preferred embodiment, this invention further provides a means andmethod for measuring antibody mediated neutralization of HIV-1 tomonitor the initial therapeutic antibody response following vaccination.

In a preferred embodiment, this invention further provides a means andmethod for measuring antibody mediated neutralization of HIV-1 over timeto monitor the durability of a protective antibody response followingvaccination.

In a preferred embodiment, this invention further provides a means andmethod for measuring antibody mediated neutralization of HIV-1 todevelop and optimize vaccination prime-boost schedules that maximizevaccination potency and durability.

For example, in the case of HIV-1, the SU protein (gp120-SU) is tightlyassociated with the transmembrane envelope protein (gp41-TM) thatanchors the complex to the virus membrane. The envelope proteins gp120and gp41 are derived by cleavage of gp160, the uncleaved precursorproduct of the envelope gene. The binding of HIV-1 to its cellularreceptor (CD4) and co-receptor (either CCR5 or CXCR4) promotesconformational changes in the TM protein resulting in the fusion of theviral and cellular membrane and entry of the virus core into thecytoplasm (Retroviruses, 1997). Although the new HIV entry inhibitorstarget either viral envelope proteins (gp120/gp41) or host proteins(CD4, CCR5, CXCR4), the majority of resistance-associated mutations inHIV-1 are expected to be located in the viral envelope gene; e.g., onelikely way viruses might evolve is to shift co-receptor utilization.Entry blockers constitute a novel class of anti-retroviral drugs, andthe potential for broad activity against current multi-drug resistantHIV-1 variants is high. Among the class of potential viral entryblockers are fusion inhibitors, receptor/co-receptor antagonists andvaccines.

Nonetheless, inhibitors of viral entry are likely to generate drugresistant viruses (through mutation of the envelope gene), thuscomplicating patient treatment similar to that observed for proteaseinhibitor (PRI) and reverse transcriptase inhibitor (RTI) treatment forHIV. In fact, FDA approval of any new drug that blocks viral entry willrequire the evaluation of resistance data. The need for a diagnosticassay that measures susceptibility to entry blockers has been documentedin the case of the fusion inhibitor T-20. Viruses exhibiting reducedsusceptibility to T-20 have been reported after passage in vitro in thepresence of the drug. At this time, convenient phenotypic assays thatare capable of measuring susceptibility to drugs that block viral entryare not available. Consequently physicians will soon be faced with thechallenge of tailoring therapy in the absence of the tools necessary toaddress drug susceptibility. Therefore, a reliable assay that accuratelymeasures susceptibility to drugs that inhibit viral entry from infectedpatients would be extremely valuable.

For example, recent World Health Organization estimates indicate thatworldwide more than 33 million people are infected with HIV-1, thecausative agent for the AIDS pandemic. Nearly one million people areinfected in the United States and 300,000 are currently receivinganti-viral therapy (CDC, 1999; WHO 1999). Combating AIDS has become thecommon goal of an unprecedented effort of governmental agencies,academic laboratories, and the pharmaceutical/biotechnology industry.Fourteen anti-viral drugs have been approved by the FDA for treatment ofHIV-1 infection (carpenter et al., 2000) and more than 20 additionaldrugs are currently being evaluated in clinical trials (PHRMA, 1999).The approved drugs inhibit HIV-1 replication by interfering with theenzymatic activities of either protease (PR) or reverse transcriptase(RT). PR inhibitors (PRIs) block the proper formation of viral proteinsthat are necessary for virus infection and replication, while RTinhibitors (RTIs) block the virus from copying its genetic material. Dueto sub-optimal potency, current PR'S and RTIs are most often used incombination to suppress viral replication (Carpenter et al., 2000).

What is desired, therefore, is to provide a rapid, accurate safe viralassay capable of evaluating: the activity of inhibitors of viralattachment and entry (including fusion, receptor and co-receptorinhibitors); receptor/co-receptor viral tropism to facilitate viralentry inhibitor drug design and treatment; changes in drugsusceptibility of patient viruses to inhibitors of attachment and entry;and viral neutralizing activity generated in response to vaccinationusing viral envelope protein antigens.

The methods of this invention can be used for any viral disease that maybe responsive to a viral entry inhibitor and where anti-viral drugsusceptibility and resistance to a viral entry inhibitor is a concernincluding, for example, including, but not limited to other lentiviruses(e.g., HIV-2), other retroviruses (e.g., HTLV-1 and 2), hepadnaviruses(e.g., human hepatitis B virus), flaviviruses (e.g., human hepatitis Cvirus) and herpesviruses (e.g., human cytomegalovirus).

Entry blockers constitute a novel class of anti-retroviral drugs, andthe potential for broad activity against current multi-drug resistantHIV-1 variants is high. Among the class of potential viral entryblockers are fusion inhibitors, receptor/co-receptor antagonists andvaccines.

Fusion Inhibitors

Compounds designed to competitively inhibit the conformational change ofTM, designated fusion inhibitors, are potent inhibitors of HIV-1replication. Although their activity has been demonstrated both in cellculture systems and HIV-1 infected patients (Wild et al., 1992; Judiceet al., 1997; Kilby et al., 1998), no fusion inhibitor has yet beenapproved for the treatment of HIV-1 infection in the U.S. Drugs withinthis class, such as T-20 and T-1249 (Trimeris Inc., USA), are thesubject of advanced clinical investigations.

Receptor/Co-receptor Antagonists

In addition to fusion inhibitors, which act after HIV-1 has interactedwith its receptors, efforts are in progress to develop drugs thatprevent HIV-1 from interacting with CD4 or either of its two principalco-receptors. The ability of such reagents to inhibit HIV-1 infectionhas been demonstrated in cell culture systems and animal models. Leadcompounds targeting either gp120, CD4, the CCR5 co-receptor used bymacrophage tropic viruses (R5), or the CXCR4 co-receptor used by T-celltropic viruses (X4) have been identified (Allaway et al., 1993; Reimannet al., 1995; Baba et al., 1999; Bridger et al., 1999).

Currently, no co-receptor antagonists are approved for the treatment ofHIV-1 infection in the U.S. Drugs within these classes, such as PRO 542(Progenies Inc., USA), 5a8 (Tanox, USA), TAK-779 (Takeda Inc., Japan),and AMID-3100 (Anormed Inc., Canada), are the subjects of preclinical orearly stage clinical investigations. Therefore, an assay capable ofidentifying and determining receptor/co-receptor tropism, which quicklyand accurately identifies patients that are infected with strains of atropic virus (e.g., HIV-1), would facilitate viral entry inhibitor drugdesign and treatment.

Vaccines

Vaccines have also proven to be an effective strategy in the fightagainst pathogenic viral infections in humans, and several vaccinecandidates to prevent HIV-I infection are in clinical development. Theenvelope proteins gp120 and gp41 are the most obvious candidates in theintense search for an HIV-1 vaccine, and many of the 11 vaccinecandidates in clinical evaluation are envelope-based (PhRMA, 1999). Itis generally thought that an effective envelope vaccine may elicit thegeneration of neutralizing antibodies that block viral infection(Mascola et al., 2000). Therefore, a sensitive high-throughput assaythat reliably measures the efficacy of such neutralizing antibodies anddoes not require prolonged cultivation of virus is urgently needed. Suchan assay could significantly aid the search for an effective AIDSvaccine. This is particularly true, considering that late-stage clinicaltrials encompass large patient populations numbering in the thousands.Since neutralizing antibodies should prevent successful infection oftarget cells, a envelope receptor assay would be beneficial to serve asa virus neutralization assay.

Unfortunately, most of these drug combinations are effective for only alimited time in large part due to the emergence of drug resistantviruses. The lack of proofreading functions inherent to RT and RNApolymerase II, coupled with high level, error-prone replication allowsviruses such as HIV-1 to mutate readily (Coffin, 1995). This highmutation frequency contributes to the ability of HIV-1 to evadesuccessful long-term drug therapy, resulting in viral load rebound.Resistance-associated mutations to all of the 14 approved drugs as wellas to many investigational compounds have been described (Schinazi etal., 1999). Consequently, multi-drug resistant HIV-1 variants pose anincreasing problem in the care of infected patients. To achievelong-term clinical benefit, it is desirable to select those drugs thatmaximally suppress viral replication and avoid the drugs to which apatient's virus is resistant (DHHs, 2000). Long-term solutions can relyon drug resistance tests that can guide physicians in selecting the mosteffective drugs against the patient's virus. The need for resistancetesting has been affirmed in recent guidelines from the DHHs (DHHs,2000), recommending that resistance tests be routinely used whentreating HIV-1 infected patients. Susceptibility tests can also assistin the development of new drugs that target resistant viruses. A recentFDA advisory committee (November 1999) recommended that resistancetesting be used in the development of new anti-viral drugs for HIV-1.

Several strategies have been applied to the assessment of antiviral drugsusceptibility. Genotypic tests analyze mutations in the underlyingnucleotide sequence, or genotype, and attempt to correlate thesemutations with drug resistance (Rodriguez-Rosado et al., 1999; Shinaziet al., 1999). However, the relationship between genotype and phenotypeis complex and not easily interpreted, and the results of these testsare not quantitative. The use of genotypic drug susceptibility datarequires interpretation either by experts (Baxter et al., 1999) orcomputer algorithms and are not always predictive of treatment outcome(Piketty et al., 1999).

Phenotypic drug susceptibility assays directly measure and quantify theability of viruses to replicate in the presence of drug. Earlyphenotypic tests required prolonged virus cultivation and consequentlywere slow, labor intensive, and not easily automated for high throughput(Japour et al., 1993). As a result, these early phenotypic tests wereconsidered impractical for patient management. The development ofrecombinant virus-assays (Shi and Mellors, 1997; Hertogs et al., 1998)simplified phenotypic testing and increased throughput. However, a majordisadvantage of these assays is a lengthy turnaround time of 4-8 weeks.More recently, recombinant virus assays have been developed and othersthat are capable of measuring drug susceptibility during a single roundof replication (Zennou et al., 1998; Petropoulos et al., 2000),resulting in a dramatic reduction in turnaround time to 8-10 days.Patients failing antiretroviral therapy can benefit from phenotypicassays. Such assays are attractive tools for patient management becausethey provide a direct and rapid measure of drug susceptibility.

The assay of this invention can be used with other viral infectionsarising from infections due to other viruses within these families aswell as viral infections arising from viruses in other viral families.In addition, the drug susceptibility and resistance test of thisinvention is useful for screening for compounds to treat viral diseasesfor which there is no currently available therapy.

The structure, life cycle and genetic elements of the viruses whichcould be tested in the drug susceptibility and resistance test of thisinvention would be known to one of ordinary skill in the art. It isuseful to the practice of this invention, for example, to understand thelife cycle of a retrovirus, as well as the viral genes required forretrovirus rescue and infectivity. Retrovirally infected cells shed amembrane virus containing a diploid RNA genome. The virus, studded withan envelope glycoprotein (which serves to determine the host range ofinfectivity), attaches to a cellular receptor in the plasma membrane ofthe cell to be infected. After receptor binding, the virus isinternalized and uncoated as it passes through the cytoplasm of the hostcell. Either on its way to the nucleus or in the nucleus, the reversetranscriptase molecules resident in the viral core drive the synthesisof the double-stranded DNA provirus, a synthesis that is primed by thebinding of a tRNA molecule to the genomic viral RNA. The double-strandedDNA provirus is subsequently integrated in the genome of the host cell,where it can serve as a transcriptional template for both mRNAs encodingviral proteins and virion genomic RNA, which will be packaged into viralcore particles. On their way out of the infected cell, core particlesmove through the cytoplasm, attach to the inside of the plasma membraneof the newly infected cell, and bud, taking with them tracts of membranecontaining the virally encoded envelope glycoprotein gene product. Thiscycle of infection—reverse transcription, transcription, translation,virion assembly, and budding—repeats itself over and over again asinfection spreads.

The viral RNA and, as a result, the proviral DNA encode severalcis-acting elements that are vital to the successful completion of theviral lifecycle. The virion RNA carries the viral promoter at its 3′end. Replicative acrobatics place the viral promoter at the 5′ end ofthe proviral genome as the genome is reverse transcribed. Just 3′ to the5′ retroviral LTR lies the viral packaging site. The retrovirallifecycle requires the presence of virally encoded transacting factors.The viral-RNA-dependent DNA polymerase (pol)—reverse transcriptase isalso contained within the viral core and is vital to the viral lifecycle in that it is responsible for the conversion of the genomic RNA tothe integrative intermediate proviral DNA. The viral envelopeglycoprotein, env, is required for viral attachment to the uninfectedcell and for viral spread. There are also transcriptionaltrans-activating factors, so called transactivators, that can serve tomodulate the level of transcription of the integrated parental provirus.Typically, replication-competent (non-defective) viruses areself-contained in that they encode all of these trans-acting factors.Their defective counterparts are not self-contained.

In the case of a DNA virus, such as a hepadnavirus, understanding thelife cycle and viral genes required for infection is useful to thepractice of this invention. The process of HBV entry has not been welldefined. Replication of HBV uses an RNA intermediate template. In theinfected cell the first step in replication is the conversion of theasymmetric relaxed circle DNA (rc-DNA) to covalently closed circle DNA(cccDNA). This process, which occurs within the nucleus of infectedliver cells, involves completion of the DNA positive-strand synthesisand ligation of the DNA ends. In the second step, the cccDNA istranscribed by the host RNA polymerase to generate a 3.5 kB RNA template(the pregenome). This pregenome is complexed with protein in the viralcore. The third step involves the synthesis of the first negativesenseDNA strand by copying the pregenomic RNA using the virally encoded Pprotein reverse transcriptase. The P protein also serves as the minusstrand DNA primer. Finally, the synthesis of the second positive-senseDNA strand occurs by copying the first DNA strand, using the P proteinDNA polymerase activity and an oligomer of viral RNA as primer. Thepregenome also transcribes mRNA for the major structural core proteins.

Design and Methods

-   -   (1) Construction of an Expression Vector for a Viral Envelope        Protein That Is Capable of Accepting Patient-derived Segments        Encoding the Envelope Protein

In one embodiment, an envelope expression vector capable of expressingHIV-1 envelope proteins in transfected cells was constructed. Similarexpression vectors have been described, including a plasmid (pAmphoEnv)constructed to express amphotropic murine leukemia virus (A-MLV)envelope protein as described in U.S. Pat. No. 5,837,464 and(Petropoulos et al., 2000). The pAmphoEnv vector uses the immediateearly gene promoter of human cytomegalovirus (CMV) and the SV40polyadenlyation signal sequence to produce A-MLV envelope mRNA intransfected cells. The pAmphoEnv plasmid is modified by deleting theA-MLV envelope gene and introducing restriction enzyme cleavage sitesthat can enable the insertion of viral envelope fragments derived from avariety of isolates, such as HIV-1. In the case of, HIV-1, the envelopeopen reading frame spans approximately 2,600 nucleotides and encodes theenvelope polyprotein, gp160. The gp160 polyprotein is cleaved by acellular furin-like protease to produce two subunits, gp41 and gp120.HIV-1 envelope expression vectors can be constructed in stages asfollows:

-   -   (a) Replacing the A-MLV Envelope Nucleic Acid Sequences from the        Envelope Expression Vector (pAmphoEnv) with a Multiple Cloning        Site Polylinker

The A-MLV envelope nucleic acid sequences can be deleted from thepAmphoEnv vector by restriction enzyme digestion. The digested vectorcan be re-circularized by ligation to a duplex oligonucleotidepolylinker containing four unique internal restriction sites (a, b, c,d) for insertion of envelope sequences. The ligation reaction can beused to transform Escherichia Coli and molecular clones containing thecorrect polylinker sequence can be identified and confirmed byrestriction mapping and DNA sequencing, respectively. The introductionof multiple unique cloning sites into the vector can facilitate theinsertion of HIV-1 envelope sequences. Restriction sites within thepolylinker can be chosen based on their infrequent occurrence in HIV-1envelope sequences (LANL HIV-1 database, www.lanl.gov). This vector canbe referred to as pCX. The functionality of the PCX vector can bedemonstrated by inserting a reporter gene or indicator nucleic acid,such as firefly luciferase, into the pCX multiple cloning site andmeasuring a signal from the indicator nucleic acid or reporter geneactivity in transfected cells. As used herein, “indicator nucleic acid”refers to a nucleic acid encoding a protein, DNA or RNA structure thateither directly or through a reaction gives rise to a measurable ornoticeable signal, e.g., color or light of measurable wavelength, orgeneration of a specific DNA or RNA structure used as an indicator whichcould be amplified using any one of a number of quantitativeamplification assays.

-   -   (b) Inserting Viral Envelope Sequences into the pCX Envelope        Expression Vector

Using mutagenic primers for PCR amplification, viral envelope fragmentsare generated that contain two unique restriction sites (a, b and c, d,respectively) adjacent to the initiation and termination codons of, forexample, the HIV-1 envelope open reading frame. Introduction of twounique restriction sites at each end of the envelope open reading framecan improve chances of cloning HIV-1 envelope fragments harboringinternal restriction sites for any one of the enzymes found in themultiple cloning site of the pCX vector.

In the case of HIV-1, two well-characterized molecular clones of HIV-1with known differences in the envelope gene, NL4-3 (asyncytium-inducing, T-cell tropic, laboratory strain) and JR-CSF (anon-syncytium-inducing, macrophage-tropic, primary isolate) can be usedas-template for PCR amplification. The 2,600 nucleotide amplificationproducts can be digested with two restriction enzymes (each enzymecleaving at one end of the fragment; e.g., a and c or b and d) andsubsequently inserted into the pCX vector by ligation and transformationof Escherichia Coli. Molecular clones containing the appropriateenvelope sequences can be identified by restriction mapping andconfirmed by DNA sequencing. The resulting plasmids, pHIVenv (NL4-3) andpHIVenv (JR-CSF), can be used to express HIV-1 envelope proteins intransfected cells (FIG. 1A). The functionality of the envelopeexpression vectors, such as the pHIVenv vectors, can be demonstrated bymeasuring viral envelope synthesis in transfected cells (Western Blot),and by their ability to pseudotype envelope deficient retrovirusvectors. High titer virus stocks using the human embryonic kidney 293cell line has been demonstrated (Petropoulos et al., 2000), however thepresent invention is not restricted to those cell lines. Other suitablecell lines used as a first cell for transfection of nucleic acidobtained from the patient encoding a viral envelope protein include, byway an example and not as limitation to the present invention, 5.25;HOX; U87; MT2; PM1; CEM; etc. The cell line optimally will be engineeredto express one or more co-receptors.

-   -   (c) Modifying the pCX hector to Improve the Efficiency of        Cloning Viral Envelope Sequences

To improve the cloning efficiency of viral envelope fragments, the PCXexpression vector can be modified by inserting a bacterial killer genecassette (e.g., control of cell death b gene (ccdB) or a member of thehok-killer gene family) under the control of the Escherichia Coli lacpromoter into the multiple cloning site (Gerdes et al, 1990; Bernard andCouturier, 1992; Bernard et al, 1993). This modified vector is referredto as pCXccdB. Transcription of the ccdB killer gene is repressed inbacterial strains that express the laci repressor, such as JM109. Thisor an equivalent strain can be used to propagate plasmids carrying theccdB killer gene that are under the control of the lac promoter.Conversely, in this system bacterial strains that do not over expressthe laciq repressor, such as DH5a® and Top10, cannot maintain plasmidsthat express the ccdB gene. Transformants can be killed due to the ccdBactivity. DH5a® and Top10 cells can be purchased from several vendors(Life Technologies or Invitrogen). Using this selective cloningapproach, the parental expression vector is propagated in a laciqbacterial strain. The vector is digested with two restriction enzymesthat both remove the ccdB gene cassette, and, in the case of HTV-1, arecompatible with the insertion of HTV-1 envelope sequences (a, b, c, d).Following ligation of the vector and envelope fragments, a strain ofbacteria lacking laciq is transformed. Once transformed,bacteria-containing plasmids in which the viral envelope inserts havereplaced the ccdB killer gene can grow. Bacteria containing plasmidsthat retain or reconstitute the ccdB killer gene can not survive. Inthis way, the population of transformed bacteria is enriched forplasmids that contain viral envelope inserts, but is lacking in theparental vector containing the ccdB gene. The construction of thepCXccdB vector is not essential for the success of phase I of thisproject, but it is expected to significantly improve the efficiency ofcloning HTV-1 envelope sequences derived from patient samples; thus, theprobability of maintaining the heterogeneity of viral sequences can beimproved. The structure of the pCXccdB vector can be confirmed byrestriction mapping and DNA sequencing.

-   -   (d) Inserting Viral Envelope Sequences into the pCXccdB        Expression Vector

The functionality of the pCXccdB vector can be evaluated by setting upligation reactions containing viral envelope sequences and incompletelydigested pCXccdB vector DNA. Following bacterial transformation, plasmidDNA can be prepared from individual bacterial clones and analyzed byrestriction digestion for the presence of viral envelope fragments andthe absence of ccdB sequences. The feasibility of this approach istested by amplifying the envelope region from a total of 13 availableHIV-1 clones (pCRII-91U5005.11, pCRII-91006.10, pCRII-92U5657.1,pCRII-92U5711.14, pCRII91US712.4, pCRII-92U5714.1, pcRII-91HT652.11,pCRII-92BR020.4, pCRII-91HT651. 1A, pCRII-92HT593.1, pCRII-92HT594.10,pCRII92HT596.4, pCRII-92HT599.24), obtainable through the AIDS researchreagent reference program (ARRRP), Rockville, Md. Each fragment can beinserted into pCXccdB and the structure of the resulting pHIVenvexpression vectors can be confirmed by restriction mapping and/or DNAsequencing. The functionality of each pHIVenv vector can be demonstratedby measuring HIV-1 envelope protein synthesis in transfected cells(Western Blot), and by their ability to pseudotype envelope-deficientretrovirus vectors.

-   -   (2) Construction of a Bio-safe Viral Expression Vector        Comprising Indicator Nucleic Acid in Place of the Encoding the        Envelope Protein

A bio-safe viral vector is constructed to evaluate inhibitors of viralentry according to similar means and methods as described in U.S. Pat.No. 5,837,464 and Petropoulos et al., 2000 used to evaluate inhibitorsof PR. and RT. The viral expression vector of the present invention canbe co-transfected into cells together with the envelope expressionvectors (described above) to produce high titer virus stocks. Such virusstocks can be evaluated for susceptibility to inhibitors of virus entry,including antiviral drugs and neutralizing antibodies. In the case ofHIV-1, the viral expression vector can be generated from NL4-3, awell-characterized infectious molecular clone of HIV-1. The 5′ longterminal repeat (LTR) which controls viral gene expression can bemodified so that transcription of the viral genes in transfected cellsis driven by the CMV immediate early promoter (Naviaux et al., 1996).Most of the envelope gene can be deleted, but important control elementssuch as the rev responsive element (RRE) and accessory protein codingregions, (rev, tat) are retained. In place of the deleted envelopesequences, an indicator nucleic acid, such as a firefly luciferasereporter gene cassette that is under the control of CMVpromoter-enhancer sequences (FIGS. 1B and 3) is inserted. Virusinfection can be monitored by measuring a luciferase activity ininfected cells. It is conceivable, although unlikely, that inter-plasmidrecombination between the retroviral vector and, for example, thepHIVenv sequences in transfected cells may lead to the generation ofinfectious HIV-1. In effort to generate a biosafe vector, introductionof several genetic alterations in the HIV genome can be done. Forexample, deletion of most of the envelope gene, while retaining theimportant control sequence, RRE, and also deletion of thetranscriptional enhancer sequences in the U3 region of the 3′ LTR of thevector (FIG. 2) can be accomplished. During the replication of theretroviral genome, the U3 region located at the 3′ end of the virusgenome serves as the template for the U3 region of the 5′ LTR of theprovirus in infected cells. Such proviruses lack the strong promoterelement in the U3 region of the 5′ LTR and thus are unable to produceretroviral RNA in infected cells. This self-inactivating (SIN) strategyhas been used successfully for several retroviral vector systems,including HIV-1 (Hwang et al., 1997; Miyoshi et al., 1998). In the assayof the present invention, viral gene expression is not required ininfected cells because virus infection is measured by a detectablesignal produced by the indicator nucleic acid, such as the production ofluciferase activity, driven by its own separate promoter (FIG. 1B).Deletion of envelope sequences and the transcriptional enhancer region(U3) can be accomplished by standard molecular cloning procedures, andeach deletion can be verified by DNA sequence analysis.

Functionality of this vector, for example in the case of HTV-1,designated pHTVlucΔU3, can be demonstrated by co-transfection of 293cells with the pHTVenv vector described above. Efficienttranscomplementation of viral proteins produced by both vectors in thetransfected cells can lead to the production of viral particles. Virusparticles can be harvested from the culture supernatants and analyzed byWestern-blotting. Virus titers can be quantitated by routineapplications of either p24 ELISA, quantitative PCR or TAQMAN® assays.

It is not necessary to produce a self-inactivating viral expressionvector to carry out the present invention, but it is desirable toimprove assay reproducibility and biosafety.

-   -   (3) Identification of Suitable Cell Lines Which Express        Receptors and Co-receptors and Support Viral Infection

Different mammalian cell lines that have been described previously andare known to support infection of a particular virus can be evaluated.As discussed herein for one embodiment relating to HIV-1, the assay canbe performed by (a) co-transfecting a first cell with pHIVenv andpHIVlucΔU3, (b) harvesting virus after transfection, (c) using thisvirus to infect a second cell, both in the presence and absence of virusentry inhibitors, and (d) measuring luciferase production in infectedcells.

Table 1 lists representative examples of such cell lines evaluated forHIV-1 infection, including the cell line and its associatedreceptor/co-receptor. Several of these cell lines can be obtained frompublic cell repositories.

Viral particles harvested from transfected 293 cell cultures can be usedto infect a variety of different cell lines. In the case of HIV-1, thepHIVlucΔU3 vector contains deletions in the envelope gene and the U3promoter-enhancer as described above, therefore infection of apermissive cell line with virus particles produced by this vector isrestricted to a single round of replication. This includes (a) virusattachment and entry, mediated by the viral envelope proteins, producedin trans by the pHIVenv vector as described, (b) the conversion ofsingle stranded viral RNA into double stranded DNA by RT, and (c)integration of viral DNA into the host cell genome (provirus formation).The active transcription of viral genes by RNA polymerase II thatnormally occurs in infected cells following proviral integration can berestricted by deleting essential viral promoter-enhancer sequences inthe pHIVlucΔU3 vector. However, this restriction cannot interfere withluciferase gene expression in infected cells since this gene is drivenindependently of viral gene expression using an internal CMV promoter(FIG. 1B). The amount of luciferase activity produced followinginfection can be used as a measure of viral infectivity.

HIV-1 attachment and entry into host cells requires interaction with aprimary receptor (CD4) and one of several co-receptors, most often CCR5or CXCR4. Cell lines can be screened that are known to express variouscombinations of CD4, CCR5 and CXCR4. Specifically, cell lines listed inTable 1 that express (a) CD4 plus CCR5, (b) CD4 plus CXCR4, and (c) CD4plus CCR5 plus CXCR4 are evaluated. Cell lines that express the CD4receptor alone, or either the CCR5 or CXCR4 co-receptor alone, may serveas useful controls and can be used to evaluate HIV-1 isolates that donot require CD4 binding or that use co-receptors other than CCR5 andCXCR4. The principal criterion for judging cell line suitability can beinfectivity as measured by luciferase production (104-106 relative lightunits). In addition, cell lines can be evaluated based on growth rates,viability, stability and other parameters as deemed necessary. Celllines can be selected that are easy to maintain and for example, producelarge amounts of luciferase activity following infection, which can beinfected by different envelope receptor tropisms, e.g., CD4/CXCR4 andCD4/CCR5. Additional well-characterized cell lines that support, forexample, HIV replication and express the HIV-1 receptor and co-receptors(e.g., CEM-NKr-CCR5; release category a) are available through publicrepositories such as the ARRRP.

Further, cell lines can be enhanced using standard procedures, such aspromoting infection by the addition of polybrene to cells (Porter etal., 1998). For example, in the case of HIV, other potential cell linescan be identified for use with the present invention by infection withHIV-1 laboratory strains and comparing the recombinant virus infectivitytiters to those obtained with infectious HIV-1, or by transfecting cellsdirectly with the viral expression plasmids described herein, andscoring for virus production. Accumulation of viral transcripts can bechecked by using a quantitative RT-PCR assay. Cell lines suitable forother viruses can be identified in a similar manner.

The present invention can optimize assay conditions and allow forhigh-throughput testing of patient samples using automation. Samplepreparation methods can be optimized to efficiently capture viralgenomic and envelope RNAs. RT-PCR conditions can be optimized to enableamplification of patient-derived viral envelope sequences, such as HIV-1envelope sequences (−2,600 base pairs) at low viral loads (−500 copiesper ml).

-   -   (4) Demonstration of the Utility of the Assay

The utility of the assay of the present invention is demonstrated by theresults achieved from: (1) testing for dose-dependent inhibition ofviral entry in the presence of well-characterized inhibitors; and the(2) testing for dose-dependent inhibition of infection in the presenceof well-characterized HIV-1 neutralizing antibodies.

The following applications for the virus entry assay of the presentinvention were evaluated:

-   -   i) detecting inhibition of HIV-1 replication by inhibitors of        virus attachment and entry—(including fusion, receptor and        co-receptor inhibitors);    -   ii) measuring changes in susceptibility to HIV-1 attachment and        entry inhibitors; and    -   iii) detecting neutralization activity of antibodies generated        in response to vaccines targeted against HIV-1 envelope        proteins.

In a preferred embodiment, the assay can be performed by (a)co-transfecting a first cell with pHIVenv and pHIVlucΔU3 vectors, (b)harvesting virus after approximately 48 h after transfection, (c) usingthis virus to infect a second cell, both in the presence and absence ofvirus entry inhibitors and (d) measuring luciferase productionapproximately 48-72 hrs. after infection. Dose-dependent inhibition ofHIV-1 replication can be evaluated against a wide range of virus entryinhibitor concentrations using a 96-well format. The appropriateconcentration range can be determined empirically for each inhibitor.The data can be plotted as the percent inhibition of luciferase activityvs. drug concentration (log10). Data analysis can be performed usingcomputer software. Inhibition curves are used to determine 50%inhibitory concentrations (IC50) for specific drugs or antibodies (FIG.6).

Envelope proteins derived from a variety of well-characterized HIV-1isolates are evaluated using pHIVenv vectors constructed as describedabove. To define envelope co-receptor tropism, in the case of HIV-1,infection using cells expressing CD4 plus CXCR4 and CD4 plus CCR5 isevaluated as described above. A wide variety of compounds that are knownto inhibit HIV-1 entry (Table 2), including non-specific agents such assulfonated polyanions (dextran sulfate and heparin) can be used with theassay of the present invention. Chemokines such as Rantes and SDF-1, thenatural ligands for the CCR5 and CXCR4 chemokine receptors, respectively(see Alkhatib et al., 1996; Bleul et al., 1996) are also suitable foruse with the present invention. Further, virus entry inhibitors such asT-20 and T1249 (Trimeris, Inc.), PRO 542 (Progenies), 5a8 (Tanox) wereused to evaluate utility of the assay of the present invention.

Drug toxicity in target cells are evaluated using standard viability orcytotoxicity assays (e.g., dye exclusion, MTS, ATP).

HIV-1 mutants exhibiting reduced susceptibility to the fusion inhibitorT20 (Rimsky et al., 1998) and the genetic determinants (mutations) thatenable these viruses to replicate in the presence of drug map within theenvelope protein (gp41-TM) have been described. To demonstrate that theassay of the present invention is capable of measuring changes in drugsusceptibility (i.e. resistance), (a) pHIVenv vectors are generated thatcarry these mutant envelope genes, (b) first cells are co-transfectedusing these vectors and the pHIVlucΔU3 vector, (c) viruses bearing thesemutant envelope proteins are harvested, and (d) the viruses are testedfor infectivity in the presence of T20. Reduced drug susceptibility toT20 is evaluated by comparing the IC50 of viruses bearing mutantenveloped proteins to those that lack the defined drug resistancemutations. Viruses bearing envelope proteins with drug resistancemutations can exhibit higher IC50 values than viruses bearing envelopeproteins that lack drug resistance mutations, i.e. inhibition canrequire a higher drug concentration (equivalent to data presented inFIG. 8). Drug resistance mutations can be introduced into envelopeexpression vectors (pHIVenv) using standard site directed mutagenesistechniques according to standard protocols (Petropoulos et al., 2000;Ziermann et al., 2000).

It is widely accepted that effective vaccines that protect against HIV-1infection should elicit a strong humoral immune response characterizedby broadly cross-reactive neutralizing antibodies. Consequently, theserum of vaccinated individuals is routinely evaluated for the presenceof high titer neutralizing antibodies targeted against the immunogen.Most recently, using the HIV-1/simian immunodeficiency virus (SIV)chimeric virus macaque model (SHIV), Mascola and colleagues have shownthat passive transfer of such neutralizing antibodies led to reducedviral load after mucosal challenge (Mascola et al., 2000). The assay ofthe present invention can be used to rapidly and reliably determine theviral neutralizing activity of antibodies generated in response tovaccines targeting envelope antigens, such HIV-1 envelope antigens. Forexample, the assay of the present invention can (a) generate pHIVenvvectors that express a variety of well-characterized envelope proteins,(b) co-transfect a first cell using these vectors and the pHIVlucΔU3vector, (c) harvest viruses and incubate with serial dilutions ofantibody preparations or vaccine serum (d) test these viruses forinfectivity in a second cell. Data analysis and IC50 determinations canbe performed as described previously and in the literature. In the caseof HIV-1, viruses can be selected to represent different HIV-1 geneticbackgrounds (e.g., Glade A, B, C, D, E, F), different cell andco-receptor tropisms (macrophage/CCR5, T-cell/CXCR4), and differentenvelope properties (syncytium and non-syncytium inducing, laboratoryadapted growth or primary isolate) (Table 2). It can be beneficial toprepare stocks of a defined titer from each virus to optimize assaysensitivity and reproducibility by using a virus input of approximately20-100 TCID₅₀/well and making adjustments as necessary. Antibodypreparations can be selected based on previously documentedneutralization properties, either functional, such as their ability toneutralize primary isolates, or physical, such as their ability to bindspecific gp120 or gp41 epitopes (Table 2). The performance of the assayof the present invention can be judged against the activity of thesewell-characterized antibody reagents in conventional virusneutralization assays as described in the scientific literature. Serumfrom a broadly representative group of HIV-1 infected individuals can beused to establish an appropriate range of serum dilutions that canmaximize assay sensitivity, yet minimize cytotoxicity. Cytoxicity can beevaluated using standard viability or cytotoxicity assays (e.g., dyeexclusion, MTS, ATP).

The following examples are presented to further illustrate and explainthe present invention and should not be taken as limiting in any regard.

Example 1: Measuring Phenotypic Drug Susceptibility to Inhibitors ofHIV-1 Entry

This example provides a means and method for accurately and reproduciblymeasuring susceptibility to inhibitors of HIV-1 attachment and entry(heretofore collectively referred to as entry). Based on this example,the means and method for measuring susceptibility to inhibitors of HIV-1entry can be adapted to other viruses, including, but not limited toother lentiviruses (e.g., HIV-2), other retroviruses (e.g., HTLV-1 and2), hepadnaviruses (human hepatitis B virus), flaviviruses (humanhepatitis C virus) and herpesviruses (human cytomegalovirus). Thisexample further provides a means and method for measuring alterations(increases and decreases) in susceptibility to entry inhibitors.

Measurements of entry inhibitor susceptibility are carried out usingadaptations of the means and methods for phenotypic drug susceptibilityand resistance tests described in U.S. Pat. No. 5,837,464 (InternationalPublication Number WO 97/27319) which is hereby incorporated byreference.

One vector, an example of the envelope expression vector, (pHIVenv) isdesigned to express the envelope polyprotein (gp160) encoded by patientderived HIV envelope sequences (FIG. 1). Gp160 is subsequently cleavedby a cellular protease to generate the surface (gp120SU) andtransmembrane (gp41TM) subunits that comprise the envelope protein onthe surface of HIV-1 virus particles. A second vector, an example of theviral expression vector, (either pHIVluc or pHIVlucΔU3) is designed toexpress genomic and subgenomic viral RNAs and all HIV proteins exceptthe envelope polyprotein (FIGS. 1A and 1B).

In this application, patient-derived segments) correspond to the codingregion (˜2.5 KB) of the HIV-1 envelope polyprotein (gp160) and representeither (a) envelope sequences amplified by the reversetranscription-polymerase chain reaction method CRT-PCR) using viral RNAisolated from virus derived from HIV-infected individuals, or (b)envelope sequences derived from molecular clones of HIV-1 that containspecific mutations introduced by site directed mutagenesis of a parentalmolecular clone (typically NL4-3).

Isolation of viral RNA was performed using standard procedures (e.g.,RNAgents Total RNA Isolation System, Promega, Madison Wis. or RNAZOL®,Tel-Test, Friendswood, Tex.). The RT-PCR protocol was divided into twosteps. A retroviral reverse transcriptase [e.g., SUPERSCRIPT® II(Invitrogen, Life Technologies) Moloney MuLV reverse transcriptase(Roche Molecular Systems, Inc., Branchburg, N.J.), or avianmyeloblastosis virus (AMV) reverse transcriptase, (Boehringer Mannheim,Indianapolis, Ind.)] was used to copy viral RNA into first strand cDNA.The cDNA was then amplified to high copy number using a thermostable DNApolymerase [e.g., Taq (Roche Molecular Systems, Inc., Branchburg, N.J.),Tth (Roche Molecular Systems, Inc., Branchburg, N.J.), PrimeZyme(isolated from Thermus brockianus, Biometra, Gottingen, Germany)] or acombination of thermostable polymerases as described for the performanceof “long PCR” (Barnes, W. M., (1994) Proc. Natl. Acad. Sci, USA 91,2216-2220) [e.g., Expand High Fidelity PCR System (Taq+Pwo), (BoehringerMannheim. Indianapolis, Ind.) or GENEAMP® XL PCR kit (Tth+Vent), (RocheMolecular Systems, Inc., Branchburg, N.J.), Advantage-2, (CloneTech).

Oligo-dT was used for reverse transcription of viral RNA into firststrand cDNA. Envelope PCR primers, forward primer Xho/Pin and reverseprimer Mlu/Xba (Table 3) were used to amplify the patient-derivedsegments. These primers are designed to amplify the ˜2.5 kB envelopegene encoding the gp160 envelope polyprotein, while introducing Xho Iand Pin AI recognition sites at the 5′ end of the PCR amplificationproduct, and Mlu I and Xba I sites at the 3′ end of the PCRamplification product.

Patient derived segments (2.5 kB envelope sequence amplificationproduct) were inserted into HIV-1 envelope expression vectors usingrestriction endonuclease digestion, DNA ligation and bacterialtransformation methods as described in U.S. Pat. No. 5,837,464(International Publication Number WO 97/27319), with minor adaptations.The about 2.5 kB amplification product was digested with either Xho I orPin AI at the 5′ end and either Mlu I or Xba I at the 3′ end. Theresulting digestion products were ligated, using DNA ligase, into the 5′Xho I/Pin AI and 3′ Mlu I/Xba I sites of modified pCXAS or pCXATexpression vectors. The construction of the pCXAS and pCXAT vectors hasbeen described in example 6 U.S. Pat. No. 5,837,464 (InternationalPublication Number WO 97/27319). Modified pCXAS and pCXAT vectorscontain a Pin AI restriction site in addition to the Xho I, MluI and XbaI restriction sites that exist in pCXAS and pCXAT. The Pin AI site wasintroduced between the Xho I and Mlu I sites by site directedmutagenesis, such that the four sites are located 5′ to 3′ in thefollowing order: Xho I, Pin AI, Mlu I and Xba I. In a preferredembodiment, the 2.5 kB amplification products were digested with Pin AIand Mlu I and ligated into the 5′ Pin AI site and the 3′ Mlu I site ofthe modified pCXAS expression vector. Ligation reaction products wereused to transform E. coli. Following a 24-36 h incubation period at30-37 C., the expression vector plasmid DNA was purified from the E.coli cultures. To ensure that expression vector preparations adequatelyrepresents the HIV quasi-species present in the serum of a givenpatient, many (>100) independent E. coli transformants were pooled andused for the preparations of pHIVenv plasmid DNA. Vectors that areassembled in this manner for the purposes of expressing patient virusderived envelope proteins are collectively referred to as pHIVenv (FIGS.1 and 3).

The genomic HIV expression vectors pHIVluc and pHIVlucΔU3 are designedto transcribe HIV genomic RNA and subgenomic mRNAs and to express allHIV proteins except the envelope polyprotein (FIG. 1B). In thesevectors, a portion of the envelope gene has been deleted to accommodatea functional indicator gene cassette, in this case, “Firefly Luciferase”that is used to monitor the ability of the virus to replicate in thepresence or absence of anti-viral drugs. In pHIVlucΔU3, a portion of the3′ U3 region has been deleted to prevent transcription of viral RNAsfrom the 5′ LTR in infected cells.

Susceptibility assays for HIV-1 entry inhibitors were performed usingpackaging host cells consisting of the human embryonic kidney cell line293 (Cell Culture Facility, UC San Francisco, SF, Calif.) and targethost cells consisting of a human osteosarcoma (HOS) cell line expressingCD4 (HT4) plus CCR5, and CXCR4, or astrocytoma (U-87) cell linesexpressing either CD4 and CCR5 or CD4 and CXCR4.

Drug susceptibility testing was performed using pHIVenv and pHIVluc orpHIVlucΔU3, Pseudotyped HIV particles containing envelope proteinsencoded by the patient derived segment were produced by transfecting apackaging host cell (HEK 293) with resistance test vector DNA. Virusparticles were collected (-48 h) after transfection and are used toinfect target cells (HT4/CCR5/CXCR4, or U-87/CD4/CXCR4, orU-87/CD4/CCR5) that express HIV receptors (i.e. CD4) and co-receptors(i.e. CXCR4, CCR5). After infection (-72 h) the target cells are lysedand luciferase activity is measured. HIV must complete one round ofreplication to successfully infect the target host cell and produceluciferase activity. The amount of luciferase activity detected in theinfected cells is used as a direct measure of “infectivity” (FIGS. 1 and2). If for any reason (e.g., lack of the appropriate receptor orco-receptor, inhibitory drug activity, neutralizing antibody binding),the virus is unable to enter the target cell, luciferase activity isdiminished. Drug susceptibility is assessed by comparing the infectivityin the absence of drug to infectivity in the presence of drug. Relativedrug susceptibility can be quantified by comparing the susceptibility ofthe “test” virus to the susceptibility of a well-characterized referencevirus (wildtype) derived from a molecular clone of HIV-1, for exampleNL4-3 or HXB2.

Packaging host cells were seeded in 10-cm-diameter dishes and weretransfected one day after plating with pHIVenv and pHIVluc orpHIVlucΔU3. Transfections were performed using a calcium-phosphateco-precipitation procedure. The cell culture media containing the DNAprecipitate was replaced with fresh medium, from one to 24 hours, aftertransfection. Cell culture media containing viral particles wastypically harvested 2 days after transfection and was passed through a0.45-mm filter. Before infection, target cells were plated in cellculture media. Entry inhibitor drugs were typically added to targetcells at the time of infection (one day prior to infection on occasion).Typically, 3 days after infection target cells were assayed forluciferase activity using the Steady-Glo reagent (Promega) and aluminometer.

In one embodiment, the susceptibility to a fusion inhibitor drug (T-20,also referred to as DP178; Trimeris, Research Triangle Park, NC) wasdemonstrated (FIG. 6). Target cells (HT4/CCR5/CXCR4) expressing CD4,CCR5 and CXCR4 were infected in the absence of T-20 and over a widerange of T-20 concentrations (x-axis log₁₀ scale). The percentinhibition of viral replication (y-axis) was determined by comparing theamount of luciferase produced in infected cells in the presence of T-20to the amount of luciferase produced in the absence of T-20. R5 tropic(JRCSF, 91US005.11), X4 tropic (NL4-3, 92HT599.24) and dual tropic(92HT593.1) viruses were tested. Drug susceptibility is quantified bydetermining the concentration of T-20 required to inhibit viralreplication by 50% (IC₅₀, shown as vertical dashed lines in FIG. 6).Viruses with lower IC₅₀ values are more susceptible to T-20 than viruseswith higher IC₅₀ values.

In still further embodiments, susceptibility to a wide variety of entryinhibitors can be measured. These inhibitors include, but are notlimited to, the drugs and compound listed in Table 4 (anti-HIV drugtable).

In a second embodiment, susceptibility to a CCR5 inhibitor belonging tothe 4-(piperidin-1-yl) butane class of compounds (Dorn, C. P. et al.,(2001), Finke, P. E. et al., (2001); Merck, West Point, Pa.) isdemonstrated. Target cells (U87/CD4/CCR5) expressing CD4 and CCR5 (R5cells) were infected in the absence of the CCR5 inhibitor and over awide range of CCR5 inhibitor concentrations (x-axis log₁₀ scale). Thepercent inhibition of viral replication (y-axis) was determined bycomparing the amount of luciferase produced in infected cells in thepresence of CCR5 inhibitor to the amount of luciferase produced in theabsence of CCR5 inhibitor. R5 tropic (JRCSF), X4 tropic (NL4-3) and dualtropic viruses (92HT593.1) were tested. Drug susceptibility wasquantified by determining the concentration of CCR5 inhibitor requiredto viral replication by 50% (IC₅₀, shown as vertical dashed lines inFIG. 8). Viruses with lower IC₅₀ values are more susceptible to the CCR5inhibitor than viruses with higher IC₅₀ values. The X4 tropic virus didnot infect the U-87/CD4/CCR5 target cells.

In a third embodiment, susceptibility to a CXCR4 inhibitor (AMD3100;AnorMED) was demonstrated. Target cells (U87/CD4/CXCR4) expressing CD4and CXCR4 were infected in the absence of the CXCR4 inhibitor and over awide range of CXCR4 inhibitor concentrations (x-axis log₁₀ scale). Thepercent inhibition of viral replication (y-axis) was determined bycomparing the amount of luciferase produced in infected cells in thepresence of CXCR4 inhibitor to the amount of luciferase produced in theabsence of CXCR4 inhibitor. R5 tropic (JRCSF), X4 tropic (NL4-3) anddual tropic (92HT593.1) viruses were tested. Drug susceptibility isquantified by determining the concentration of CXCR4 inhibitor requiredto inhibit viral replication by 50% (IC₅₀, shown as vertical dashedlines in FIG. 9). Viruses with lower IC₅₀ values are more susceptible tothe CCR5 inhibitor than viruses with higher IC₅₀ values. The R5 tropicvirus did not infect the U-87/CD4/CXCR4 target cells.

Susceptibility to a CD4 inhibitor (e.g., murine monoclonal antibody 5A8;Tanox, Houston, Tex.) can be measured. Target cells (e.g.,HT4/CCR5/CXCR4, U-87/CD4/CXCR4, or U-87/CD4/CCR5) expressing CD4 and oneor both co-receptors can be infected in the absence of the CD4 inhibitordrug and over a wide range of CD4 inhibitor drug concentrations (x-axislog₁₀ scale). The percent inhibition of viral replication (y-axis) canbe determined by comparing the amount of luciferase produced in infectedcells in the presence of CD4 inhibitor to the amount of luciferaseproduced in the absence of CD4 inhibitor. R5 tropic (e.g., JRCSF), X4tropic (e.g., NL4-3) and dual tropic (e.g., 92HT593.1) viruses can betested. Drug susceptibility can be quantified by determining theconcentration of CD4 inhibitor required to inhibit viral replication by50% (IC₅₀), Viruses with lower IC₅₀ values are more susceptible to theCD4 inhibitor than viruses with higher IC₅₀ values.

Example 2: Discovery, Optimization and Characterization of New and NovelInhibitors of Virus Entry

In one embodiment, the virus entry assay can be used to identify newcompounds/chemical entities that inhibit virus entry. Target cells(e.g., HT4/CCR5/CXCR4, U-87/CD4/CXCR4, or U-37/CD4/CCR5) expressing CD4and one or both co-receptors can be infected in the presence ofindividual members of large chemical libraries (high throughputscreening, HTS). The ability of a compound to inhibit viral replication(a “hit”) can be determined by comparing the amount of luciferaseproduced in infected target cells in the presence of a specific compoundto the amount of luciferase produced in the absence of the compound.

In a further embodiment, the virus entry assay can be used to optimizethe antiviral activity of lead compounds identified by HTS. Chemicalmodified derivatives of lead compounds can be tested to identifyspecific derivatives that have enhanced virus entry inhibitory activity.Target cells (e.g., HT4/CCR5/CXCR4, U-87/CD4/CXCR4, or U-87/CD4/CCR5)expressing CD4 and one or both co-receptors can be infected in theabsence of the inhibitor candidate and over a wide range of inhibitorcandidate concentrations (x-axis log₁₀ scale). The percent inhibition ofviral replication (y-axis) can be determined by comparing the amount ofluciferasc produced in infected cells in the presence of the candidateinhibitor to the amount of luciferase produced in the absence ofcandidate inhibitor. Drug susceptibility can be quantified bydetermining the concentration of inhibitor candidate required to inhibitviral replication by 50% (IC₅₀). Derivatized compounds with lower IC₅₀values are more potent inhibitors of virus entry (have greater antiviralactivity) than derivatives with higher IC₅₀ values.

In yet a further embodiment, the virus entry assay can be used tocharacterize the mechanism of action of new virus entry inhibitor drugcandidates, and the antiviral activity against a spectrum of virusesthat may differ in susceptibility. Target cells (e.g., HT4/CCR5/CXCR4,U87/CD4/CXCR4, or U-87/CD4/CCR5) expressing CD4 and one or bothco-receptors can be infected in the absence of the new entry inhibitordrug candidate and over a wide range of entry inhibitor drugconcentrations (x-axis log₁₀ scale). The percent inhibition of viralreplication (y-axis) can be determined by comparing the amount ofluciferase produced in infected cells in the presence of new entryinhibitor to the amount of luciferase produced in the absence of the newentry inhibitor, R5 tropic (e.g., JRCSF), X4 tropic (e.g., NL4-3) anddual tropic (e.g., 92HT593.1) viruses can be tested. Drug susceptibilitycan be quantified by determining the concentration of CD4 inhibitorrequired to inhibit viral replication by 50% (IC₅₀).

To determine whether the new entry inhibitor acts by blocking the CCR5or CXCR4 co-receptors, the R5 tropic viruses are tested against the newinhibitor in U-87/CD4/CCR5 cells and X4 tropic viruses are testedagainst the new inhibitor using U-87/CD4/CXCR4 cells. Inhibition of R5virus infection is indicative of CCR5 co-receptor antagonism andconversely, inhibition of X4 virus infection is indicative of CXCR4coreceptor antagonism, Inhibition of R5 and X4 virus infection may beindicative of either CD4 antagonism or the inhibition of membranefusion.

To characterize the activity of a new inhibitor against viruses thatexhibit resistance, or have reduced susceptibility, to other virus entryinhibitors of the same class, or different class, selected panels ofdrug resistant viruses can be tested in the virus entry assay using thenew entry inhibitor drug. The panel may include viruses with varyinglevels of susceptibility to CCR5 inhibitors, CXCR4 inhibitors, CD4inhibitors, and membrane fusion inhibitors. The panel may includeviruses with one or more specific mutations that are associated withreduced susceptibility/resistance to one or more entry inhibitors.

Example 3: Identifying Envelope Amino Acid Substitutions/Mutations thatAlter Susceptibility to Virus Entry Inhibitors

This example provides a means and method for identifying mutations inHIV-1 envelope that confer reduced susceptibility/resistance to virusentry inhibitors. This example also provides a means and method forquantifying the degree of reduced susceptibility to entry inhibitorsconferred by specific envelope mutations.

Envelope sequences derived from patient samples, or individual clonesderived from patient samples, or envelope sequences engineered by sitedirected mutagenesis to contain specific mutations, are tested in theentry assay to quantify drug susceptibility based on awell-characterized reference standard (e.g., NL4-3, HXB2).

In one embodiment, susceptibility to longitudinal patient samples(viruses collected from the same patient at different timepoints) isevaluated. For example, susceptibility to entry inhibitors is measuredprior to initiating therapy, before or after changes in drug treatment,or before or after changes in virologic (RNA copy number), immunologic(CD4 T-cells), or clinical (opportunistic infection) markers of diseaseprogression.

-   Genotypic analysis of patient HIV samples

Envelope sequences representing patient sample pools, or clones derivedfrom patient pools, can be analyzed by any broadly available DNAsequencing methods. In one embodiment of the present invention, patientHIV sample sequences are determined using viral RNA purification, RT/PCRand dideoxynucleotide chain terminator sequencing chemistry andcapillary gel electrophoresis (Applied Biosystems, Foster City, Calif.).Envelope sequences of patient virus pools or clones are compared toreference sequences, other patient samples, or to a sample obtained fromthe same patient prior to initiation of therapy, if available. Thegenotype is examined for sequences that are different from the referenceor pre-treatment sequence and correlated to differences in entryinhibitor susceptibility.

-   Entry inhibitor susceptibility of site directed mutants

Genotypic changes that correlate with changes in fitness are evaluatedby constructing envelope expression vectors (pHIVenv) containing thespecific mutation on a defined, drug susceptible, genetic background(e.g., NL4-3 reference strain). Mutations may be incorporated aloneand/or in combination with other mutations that are thought to modulatethe entry inhibitor susceptibility. Envelope mutations are introducedinto pHIVenv vectors using any of the broadly available methods forsite-directed mutagenesis. In one embodiment of this invention themega-primer PCR method for site-directed mutagenesis is used (Sarkar, G.and Summer, S. S., 1990). A pHIVenv vector containing a specificenvelope mutation or group of mutations are tested using the virus entryassay described in Example 1. Drug susceptibility of the viruscontaining envelope mutations is compared to the drug susceptibility ofa genetically defined drug susceptible virus that lacks the specificmutations under evaluation. Observed changes in entry inhibitorsusceptibility are attributed to the specific mutations introduced intothe pHIVenv vector.

In one embodiment of the present invention, reduced susceptibility tothe fusion inhibitor T-20 conferred by specific drug resistancemutations in the gp41 envelope protein is demonstrated (FIG. 7). Cellsexpressing CD4, CCR5 and CXCR4 were infected in the absence of T-20 andover a wide range of T-20 concentrations (x-axis log¹⁰ scale). Thepercent inhibition of viral replication (y-axis) was determined bycomparing the amount of luciferase produced in infected cells in thepresence of T-20 to the amount of luciferase produced in the absence ofT-20. Isogenic viruses containing one or two specific mutations in thegp41 transmembrane envelope protein were tested (highlighted in red inthe figure legend; Rimsky et al., J. Virol. 72: 986-993). Drugsusceptibility is quantified by determining the concentration of T-20required to inhibit viral replication by 50% (IC⁵⁰, shown as verticaldashed lines). Viruses with lower IC⁵⁰ values are more susceptible toT-20 than viruses with higher IC⁵⁰ values.

In one embodiment, drug resistance mutations were introduced intowell-characterized X4 tropic (NL4-3) and R5 tropic (JRCSF) viruses. T-20susceptibility was measured using the virus entry assay (FIG. 7). Thefold change (FC) in T-20 susceptibility for each virus was determined bydividing the IC⁵⁰ of the test virus by the IC⁵⁰ of the HXB2 strain ofHIV-1. T-20 sensitivity of similar mutant viruses has been reported inthe scientific literature (Rimsky et al. In this embodiment, viruseswith one mutation within the GIV motif of gp41 (DIV, GIM, SIV) were lesssusceptible to T20 than the wildtype virus (GIV). Viruses with twomutations within the GIV motif (DIM, SIM, DTV) were less susceptible toT20 than viruses with one, or no mutations in the GIV motif.

In another embodiment, mutations that may confer reduced (or increased)susceptibility to the entry inhibitor are identified by sequencing theenvelope genes of the sensitive and resistant viruses. The deduced aminoacid sequences of the sensitive and resistant viruses are compared toidentify candidate drug resistance mutations. The ability of a specificmutation to confer altered drug susceptibility is confirmed or disprovedby introducing the mutation into a drug sensitive virus and measuringthe susceptibility of the mutant virus in the virus entry assay. In theexample represented here, a short stretch of amino acid sequences withinthe first heptad repeat (HR-1) of the HIV-1 gp41 transmembrane envelopeprotein is aligned for viruses exhibiting different T-20susceptibilities. Highlighted amino acids represent mutations known toconfer reduced susceptibility to T-20.

Similar phenotypic and genotypic analyses can be used to identifyenvelope amino acid sequences that (a) alter/influence susceptibility toCCR5 or CXCR4 inhibitors, (b) specify X4, R5 and dual tropism, and (c)elicit neutralizing antibodies.

In one embodiment, reduced susceptibility to co-receptor (CCR5, CXCR4)inhibitors conferred by specific envelope amino acid sequences/mutationsis demonstrated.

In a further embodiment, reduced susceptibility to receptor (CD4)inhibitors conferred by specific envelope amino acid sequences/mutationsis demonstrated.

Example 4: Determining HIV-1 Co-Receptor and Receptor Tropism

This example provides a means and method for determining HIV-1co-receptor tropism. This example also provides a means and method fordetermining HIV-1 receptor tropism.

In one embodiment, viruses that use the CCR5 co-receptor are identified.In a related embodiment, viruses that use the CXCR4 co-receptor areidentified. In a further related embodiment, viruses that use CCR5 andCXCR4 are identified. In a further related embodiment, viruses that useco-receptors other than CCR5 or CXCR4 are identified.

In another embodiment, viruses that use the CD4 receptor are identified.In a related embodiment, viruses that use CD8 are identified. In afurther related embodiment, viruses that do not require CD4 or CD8 toinfect cells are identified.

In this embodiment, the assay is performed using two cell lines. Onecell line expresses CD4 plus CCR5 (U87/CD4/CCR5), also referred to as R5cells in this application. The other cell line expresses CD4 and CXCR4(U87/CD4/CXCR4) also referred to as X4 cells in this application. Thevirus entry assay is performed by infecting individual cell cultureswith recombinant virus stocks derived from cells transfected withpHIVenv and pHIVluc or pHIVlucΔU3 vectors. pHIVenv vectors containpatient virus derived sequences and express HIV-1 envelope proteins(gp120SU, gp41TM). In this embodiment viruses are evaluated in using R5and X4 target cells cultured in 96 well plates (FIG. 3A). Typically, R5and X4 cells are plated one day prior to infection. Infection with eachvirus stock is performed in the absence of drug (no drug), in thepresence of inhibitory concentrations of a drug that preferentiallyinhibits R5 tropic viruses (CCR inhibitor, e.g., a piperidinlyl butanecompound), and in the presence of inhibitory concentrations of a drugthat preferentially inhibits X4 tropic viruses (CXCR4 inhibitor, e.g.,AMD3100). Co-receptor tropism is assessed by comparing the amount ofluciferase activity produced in each cell type, both in the presence andabsence of drug. In this embodiment, the results of the assay areinterpreted by comparing the ability of each virus to preferentiallyinfect (produce luciferase activity) R5 cells or X4 cells, or both X4and R5 cells if the virus is dual tropic. The ability of the CCR5 orCXCR4 inhibitor to specifically block infection (inhibit luciferaseactivity) is also evaluated (FIG. 3B). In this embodiment, X4 tropicviruses infect X4 cells but not R5 cells and infection of X4 cells isblocked by the CXCR4 inhibitor (AMD3100). In this embodiment, R5 tropicviruses infect R5 cells but not X4 cells and infection of R5 cells isblocked by the CCR5 inhibitor (piperidin-1yl butane compound). In thisembodiment, dual tropic, or mixtures of X4 and R5 tropic viruses, infectboth X4 and R5 cells and infection of R5 cells is blocked by the CCR5inhibitor and infection of X4 cells is blocked by the CXCR4 inhibitor.In this embodiment, non-viable viruses do not replicate in either X4 orR5 cells (luciferase activity is not produced).

In another embodiment, the assay is performed using three or more celllines. One cell line expresses CD4 plus CCR5 (U87/CD4/CCR5), alsoreferred to as R5 cells in this application. The other cell lineexpresses CD4 and CXCR4 (U87/CD4/CXCR4) also referred to as X4 cells inthis application. Additional cell lines express CD4 plus other candidateHIV-1 co-receptors, including, but not limited to, BONZO, BOB, etc. SeeTable 1. These additional cell lines express other candidateco-receptors, but do not express CCR5 or CXCR4. The virus entry assay isperformed by infecting individual cell cultures with recombinant virusstocks derived from cells transfected with pHIVenv and pHIVluc orpHIVlucΔU3 vectors. pHIVenv vectors contain patient virus derivedsequences and express HIV-1 envelope proteins (gp120SU, gp41TM). In thisembodiment viruses are evaluated in using cells cultured in 96 wellplates. Infection with each virus stock is performed in R5 cells, X4cells and the cell lines expressing CD4 plus the candidate co-receptors.Co-receptor tropism is assessed by comparing the amount of luciferaseactivity produced in each cell type. In this embodiment, the results ofthe assay are interpreted by comparing the ability of each virus topreferentially infect (produce luciferase activity) R5 cells or X4cells, or the cell line that expresses the candidate co-receptor. Inthis embodiment, X4 tropic viruses infect X4 cells but not R5 cells. Inthis embodiment, R5 tropic viruses infect R5 cells but not X4 cells. Inthis embodiment dual tropic, or mixture of X4 and R5 tropic viruses,infect both X4 and R5 cells. In this embodiment, the infection of celllines expressing alternative candidate co-receptors (neither CCR5 orCXCR4) is attributed to tropism for the alternative coreceptor. In thisembodiment, non-viable viruses do not replicate in either X4 or R5cells.

In another embodiment, the assay is performed using four cell lines. Onecell line expresses CD4 plus CCR5 (U87/CD4/CCR5), also referred to as R5cells in this application. A second other cell line expresses CD4 andCXCR4 (U-87/CD4/CXCR4) also referred to as X4 cells in this application.A third cell line expresses CD8 plus CCR5 (U87/CD8/CCR5), also referredto as CD8/R5 cells in this application. A fourth cell line expresses CD8and CXCR4 (U87/CD8/CXCR4) also referred to as CD8/X4 cells in thisapplication. The virus entry assay is performed by infecting individualcell cultures with recombinant virus stocks derived from cellstransfected with pHIVenv and pHIVluc or pHIVlucΔU3 vectors. pHIVenvvectors contain patient virus derived sequences and express HIV-1envelope proteins (gp120SU, gp41TM). In this embodiment viruses areevaluated in using cells cultured in 96 well plates. Infection with eachvirus stock is performed in R5 cells, X4 cells, CD8/R5 cells and CD8/X4cells. Co-receptor tropism is assessed by comparing the amount ofluciferase activity produced in each cell type. In this embodiment, theresults of the assay are interpreted by comparing the ability of eachvirus to preferentially infect (produce luciferase activity) R5 cells,X4 cells, CD8/R5 cells, or CD8/X4 cells. In this embodiment, CD4 tropicviruses infect X4 cells and/or R5 cells. In this embodiment, CD8 tropicviruses infect CD8/R5 cells and/or CD8/X4 cells. In this embodiment,dual tropic (CD4 and CD8 receptor use) viruses infect X4 cells and/or R5cells plus CD8/X4 and/or CD8/R5 cells. In this embodiment, the infectionof cell lines expressing CD8 but not CD4 is attributed to CD8 receptortropism. In this embodiment, non-viable viruses do not replicate ineither X4 or R5 cells.

In a further related embodiment, the assay is performed using two celllines. One cell line expresses CD4 plus CCR5 and CXCR4 (HT4/CCR5/CXCR4).A second cell line expresses CD8 plus CCR5 and CXCR4(HOS/CD8/CCR5/CXCR4). The virus entry assay is performed by infectingindividual cell cultures with recombinant virus stocks derived fromcells transfected with pHIVenv and pHIVluc or pHIVlucΔU3 vectors.pHIVenv vectors contain patient virus derived sequences and expressHIV-1 envelope proteins (gp120SU, gp41TM). In this embodiment virusesare evaluated in using cells cultured in 96 well plates. Infection witheach virus stock is performed in HT4/CCR5/CXCR4 cells andHOS/CD8/CCR5/CXCR4 cells. Co-receptor tropism is assessed by comparingthe amount of luciferase activity produced in each cell type. In thisembodiment, the results of the assay are interpreted by comparing theability of each virus to preferentially infect (produce luciferaseactivity) HT4/CCR5/CXCR4 cells or HOS/CD8/CCR5/CXCR4 cells. In thisembodiment, CD4 tropic viruses infect HT4/CCR5/CXCR4 cells, but notHOS/CDS/CCR5/CXCR4 cells. in this embodiment, CD8 tropic viruses infectHOS/CD8/CCR5/CXCR4 cells but not HT4/CCR5/CXCR4 cells. In thisembodiment, dual tropic (CD4 and CDS receptor use) viruses infect bothHT4/CCR5/CXCR4 cells and HOS/CD8/CCR5/CXCR cells. In this embodiment,the infection of cell lines expressing CD8 but not CD4 is attributed toCDS receptor tropism. In this embodiment, nonviable viruses do notreplicate in either X4 or R5 cells.

In another embodiment, the assay is performed using two cell lines. Onecell line expresses CD4 plus CCR5 and CXCR4 (HT4/CCR5/CXCR4). A secondcell line expresses CCR5 and CXCR4 but not CD4 or CD8 (HOS/CCR5/CXCR4).The virus entry assay is performed by infecting individual cell cultureswith recombinant virus stocks derived from cells transfected withpHIVenv and pHIVluc or pHIVlucΔU3 vectors. pHIVenv vectors containpatient virus derived sequences and express HIV-1 envelope proteins(gp120SU, gp41TM). In this embodiment viruses are evaluated in usingcells cultured in 96 well plates. Infection with each virus stock isperformed in HT4/CCR5/CXCR4 cells and HOS/CCR5/CXCR4 cells. CD4 and CD8independent infection is assessed by comparing the amount of luciferaseactivity produced in each cell type. In this embodiment, the results ofthe assay are interpreted by comparing the ability of each virus topreferentially infect (produce luciferase activity) HT4/CCR5/CXCR4 cellsor HOS/CCR5/CXCR4 cells. In this embodiment, CD4 dependent virusesinfect HT4/CCR5/CXCR4 cells, but not HOS/CCR5/CXCR4 cells. In thisembodiment, CD4 independent viruses infect both HOS/CCR5/CXCR4 cells andHT4/CCR5/CXCR4 cells. In this embodiment, the infection of cell linesthat lack CD4 expression is attributed to CD4 independent infection. Inthis embodiment, non-viable viruses do not replicate in either X4 or R5cells.

Example 5: Identifying HIV-1 Envelope Amino Acid Substitutions/Mutationsthat Alter Co-Receptor and Receptor Tropism

This example provides a means and method for identifying HIV1 envelopeamino acid sequences that specify, or alter, co-receptor tropism (X4 vs.R5 vs. dual X4/R5). This example also provides a means and method foridentifying HIV-1 envelope amino acid sequences that specify co-receptorusage other than CXCR4 or CCR5. The example also provides a means andmethod for identifying HIV-1 envelope sequences that specific, orreceptor tropism (CD4 vs. CD8).

Envelope sequences derived from patient samples, or individual clonesderived from patient samples, or envelope sequences engineered by sitedirected mutagenesis to contain specific mutations, are tested in theentry assay to determine co-receptor tropism as described in Example 4.

In one embodiment, co-receptor tropism of longitudinal patient samples(viruses collected from the same patient at different timepoints) isevaluated. For example, co-receptor tropism is evaluated prior toinitiating therapy, before or after changes in drug treatment, or beforeor after changes in virologic (RNA copy number), immunologic (CD4T-cells), or clinical (opportunistic infection) markers of diseaseprogression.

In another embodiment, co-receptor tropism is evaluated for samplescollected from a large number of different patients. In a furtherembodiment, co-receptor tropism is evaluated for samples collected froma large number of patients representing different virus and patientpopulations. Such patient populations may include, but are not limitedto, newly infected patients, chronically infected patients, patientswith advanced disease, and patients undergoing antiretroviral therapy orimmune-therapy. Such virus populations may include, but are not limitedto, viruses with distinct genetic characteristics (clade A, B, C, D, E,F, G), viruses susceptible to antiretroviral drugs, viruses with reducedsusceptibility/resistance to antiretroviral drugs.

-   Genotypic analysis of patient HIV samples

Envelope sequences representing patient sample pools, or clones derivedfrom patient pools, can be analyzed by any broadly available DNAsequencing methods. In one embodiment of the present invention, patientHIV sample sequences are determined using viral RNA purification, RT/PCRand dideoxynucleotide chain terminator sequencing chemistry andcapillary gel electrophoresis (Applied Biosystems, Foster City, Calif.).Envelope sequences of patient virus pools or clones are compared toreference sequences, other patient samples, or to a sample obtained fromthe same patient prior to initiation of therapy, if available. Thegenotype is examined for sequences that are different from the referenceor pre-treatment sequence and correlated to differences in entryinhibitor susceptibility.

-   Co-receptor and Receptor tropism of genetically characterized    viruses

Envelope amino acid sequences that correlate co-receptor tropism areevaluated by constructing envelope expression vectors (pHIVenv)containing a specific mutation on a defined genetic background (e.g.,NL4-3 for X4 tropism, JRCSF for R5 tropism). Mutations may beincorporated alone and/or in combination with other mutations that arethought to modulate co-receptor usage. Envelope mutations are introducedinto pHIVenv vectors using any of the broadly available methods forsite-directed mutagenesis. In one embodiment of this invention themega-primer PCR method for site-directed mutagenesis is used (Sarkar, G.and Summer, S. S., 1990). A pHIVenv vector containing a specificenvelope mutation or group of mutations are tested using the vitalsentry assay described in Example 1. Co-receptor tropism of the viruscontaining envelope mutations is compared to the co-receptor tropism ofa genetically defined virus that lacks the specific mutations underevaluation. The ability of a specific mutation to confer alteredco-receptor tropism is confirmed or disproved by introducing themutation into well-characterized reference virus and evaluating theco-receptor tropism of the mutant virus in the virus entry assay asdescribed in Example 4. Observed changes in co-receptor tropism areattributed to the specific mutations introduced into the pHIVenv vector.

In one embodiment of this invention, genetic determinants of R5 tropismare identified by evaluating amino acid sequences within the V3 loop ofthe gp120 surface envelope protein. The amino acid sequences underevaluation are identified by comparing the amino acid sequences of largenumbers of X4 tropic and R5 tropic viruses. Consistent differencesbetween the X4 and R5 viruses are selected for evaluation. Isogenicviruses based on an well-characterized X4 parental clone (e.g. NL4-3,HXB2) containing specific “R5 candidate” mutations in the V3 loop of thegp120 envelope protein are constructed by site directed mutagenesis andtested for co-receptor tropism as described in Example 4. Cellsexpressing CD4 plus CCR5 (e.g., U-87/CD4/CCR5) or CD4 plus CXCR4(U-87/CD4/CXCR4) are infected in the absence of an R5 (peperidin-1ylbutane compound) and X4 (AMD3100) inhibitor and in the presence ofinhibitory concentrations of R5 and X4 drug concentrations. Amino acidsubstitutions that change the X4 tropic virus to an R5 tropic virus arecharacterized as genetic determinants of R5 tropism.

In a related embodiment of the present invention, genetic determinantsof X4 tropism are identified by evaluating amino acid sequences withinthe V3 loop of the gp120 surface envelope protein. The amino acidsequences under evaluation are identified by comparing the amino acidsequences of large numbers of X4 tropic and R5 tropic viruses,Consistent differences between the X4 and R5 viruses are selected forevaluation. Isogenic viruses based on an well-characterized R5 parentalclone (e.g., JRCSF) containing specific “X4 candidate” mutations in theV3 loop of the gp120 envelope protein are constructed by site directedmutagenesis and tested for co-receptor tropism as described in Example4. Cells expressing CD4 plus CCR5 (e.g., U-87/CD4/CCR5) or CD4 plusCXCR4 (U-87/CD4/CXCR4) are infected in the absence of an R5(peperidin-1yl butane compound) and X4 (AMD3100) inhibitor and in thepresence of inhibitory concentrations of R5 and X4 drug concentrations.Amino acid substitutions that change the X4 tropic virus to an R5 tropicvirus are characterized as genetic determinants of R5 tropism.

In a related embodiment of this invention, genetic determinants of X4 orR5 tropism are identified by evaluating amino acid sequences within theentire gp120 surface envelope protein.

In a related embodiment of the invention, genetic determinants of X4 orR5 tropism are identified by evaluating amino acid sequences within thegp41 transmembrane envelope protein.

In a related embodiment of the invention, genetic determinants thatspecify the use of co-receptors other than CCR5 and CXCR4 are identifiedby evaluating amino acid sequences within the V3 loop of the gp120surface envelope protein. The amino acid sequences under evaluation areidentified by comparing the amino acid sequences of viruses that areable to replicate on cells that do not express CXCR4 or CCR5, but doexpress other candidate co-receptors. Consistent differences in aminoacid sequences between these non-X4, non R5 viruses and the X4 and R5viruses are selected for evaluation. Isogenic viruses based on anwell-characterized X4 (e.g., NL4-3) or R5 (e.g., JRCSF) parental clonecontaining specific “non-X4, non-R5 candidate” mutations in the V3 loopof the gp120 envelope protein are constructed by site directedmutagenesis and tested for co-receptor tropism as described in Example4. Cells expressing CD4 plus CCR5 (e.g., U-87/CD4/CCR5), CD4 plus CXCR4(U87/CD4/CXCR4), and CD4 plus other candidate co-receptors (U87/CD4/X)are infected in the absence of an R5 (peperidin-1yl butane compound) andX4 (AMD3100) inhibitor and in the presence of inhibitory concentrationsof R5 and X4 drug concentrations. Amino acid substitutions that conferstropism for a non-X4, non-R5 co-receptor are characterized as geneticdeterminants of tropism for the specific co-receptor.

In a related embodiments of the invention, genetic determinants oftropism for other co-receptors are identified by evaluating amino acidsequences within the entire gp120 surface envelope protein.

In a related embodiment of the invention, genetic determinants oftropism for other co-receptors are identified by evaluating amino acidsequences within the gp41 transmembrane envelope protein.

In another embodiment of the invention, genetic determinants thatspecify the use of CD8 (in addition to, or instead of CD4) as a receptorfor HIV-1 are identified by evaluating amino acid sequences within theV3 loop of the gp120 surface envelope protein. The amino acid sequencesunder evaluation are identified by comparing the amino acid sequences ofviruses that are able to replicate in cells that do not express CD4, butdo express CD8. Consistent differences in amino acid sequences betweenthese CD4 tropic viruses and CD8 tropic viruses are selected forevaluation. Isogenic viruses based on an well-characterized CD4 tropic(e.g., NL4-3, JRCSF) parental clones containing specific “CD8 candidate”mutations in the V3 loop of the gp120 envelope protein are constructedby site directed mutagenesis and tested for CD8 receptor tropism asdescribed in Example 4. Cells expressing CD4 plus CCR5 (e.g.,U-87/CD4/CCR5), CD4 plus CXCR4 (U-87/CD4/CXCR4), CD8 plus CCR5 (e.g.,U-87/CD8/CCR5), CD8 plus CXCR4 (U87/CD8/CXCR4) are infected. Amino acidsubstitutions that enable replication in cells that express CD8 but notCD4 are characterized as genetic determinants of CD8 tropism.

In a related embodiments of the invention, genetic determinants of CD8tropism are identified by evaluating amino acid sequences within theentire gp120 surface envelope protein.

In a related embodiment of the invention, genetic determinants of CD8tropism are identified by evaluating amino acid sequences within thegp41 transmembrane envelope protein.

Example 6: Measuring HIV-1 Antibody Neutralization

This example provides a means and method for evaluating antibodymediated neutralization of HIV-1, also referred to as virusneutralization in this application. This example also provides a meansand method for evaluating the virus neutralization activity ofantibodies within HIV-1 infected patients. This example also provides ameans and method for evaluating the virus neutralizing activity ofantibodies within individuals or animals vaccinated with therapeuticvaccines and vaccine candidates. This example also provides a means andmethod for evaluating the virus neutralizing activity of antibodieswithin individuals or animals vaccinated with protective (preventativeor prophylactic) vaccine and vaccine candidates. This example alsoprovides a means and method for evaluating the virus neutralizingactivity or preparations of specific monoclonal or polyclonalantibodies.

Envelope sequences derived from patient samples, or individual clonesderived from patient samples, or envelope sequences engineered by sitedirected mutagenesis to contain specific mutations, are tested in theentry assay to evaluate antibody mediated neutralization.

In one embodiment, antibody mediated neutralization is evaluated inlongitudinal patient samples (viruses collected from the same patient atdifferent time points) is evaluated. For example, virus neutralizationis evaluated prior to vaccination, during a course of vaccination, andat incremental time points after the course of vaccination is completed.In a related embodiment, the sera of animals including, but not limitedto, mice, rats, rabbits, pigs, and cattle, are evaluated prior toinoculation with candidate vaccines, during a course of repeatedinoculation, and at incremental time points after the course ofinoculation is completed. In one embodiment, virus neutralization isevaluated for preventative vaccines and vaccine candidates. In anotherembodiment, virus neutralization is evaluated for therapeutic vaccines.

In another embodiment, virus neutralization is evaluated for samplescollected from a large number of different patients. In a furtherembodiment, virus neutralization is evaluated for samples collected froma large number of patients representing different patient populationsand different virus populations. “Patient populations” may include, butare not limited to, newly infected patients, chronically infectedpatients, patients with advanced HIV/AIDS disease, patients with rapiddisease progression, patients with slow disease progression (typicallyreferred to as long term non-progressors), patients undergoingantiretroviral therapy or immune-therapy (e.g., interleukin-2, or othercytokines), vaccinated and unvaccinated individuals. “Virus populations”may include, but are not limited to, viruses with distinct geneticcharacteristics and geographical origins (clade-A, B, C, D, E, F, G),viruses susceptible to antiretroviral drugs, viruses with reducedsusceptibility/resistance to antiretroviral drugs, primary isolates,isolates adapted for growth in cell culture (often referred to aslab-adapted viruses), syncytia inducing (SI) viruses, non-syncytiainducing (NSI) viruses, macrophage (M) tropic viruses, T-cell (T) tropicviruses and dual tropic (M and T) viruses.

Characterization of Patient Antibody (Patient Antibody Vs. StandardVirus Panel)

In this embodiment, the assay is performed using a target cell line thatexpresses the HIV-1 receptor CD4 plus the HIV-1 co-receptors CCR5 andCXCR4 (HT4/CCR5/CXCR4). Such a cell line is capable of evaluating theneutralizing activity of antibodies for both R5 and X4 tropic viruses.In a related embodiment, the assay is performed using two target celllines. One cell line expresses CD4 plus CCR5 (U-87/CD4/CCR5) and is usedto test R5 tropic viruses. Another cell line expresses CD4 plus CXCR4(U-87/CD4/CXCR4) and is used to evaluate X4 tropic viruses. The virusentry assay is performed by infecting individual target cell cultureswith recombinant virus stocks derived from packaging host cellstransfected with pHIVenv and pHIVluc or pHIVlucΔU3 vectors. In thisembodiment, pHIVenv vectors contain envelope sequences of specific,well-characterized viruses and express the HIV-1 envelope proteins(gp120SU, gp41TM). Such viruses represent a “standard virus panel” (seeabove description of virus population). Some, but not all, reasonableexamples of viruses that may constitute a standard panel are listed inTable 4. A standard virus panel is used to compare the neutralizingantibody activity of sera obtained from many different patients and/oranimals (see above description of patient population). In thisembodiment viruses are evaluated using target cells cultured in 96 wellplates. Typically, target cells are plated at 5,000 cells per well forHT4/CCR5/CXCR4 or 10,000 cells per well for U-87/CD4/CCR5 andU-87/CD4/CXCR4 one day prior to infection. Prior to target cellinfection, each virus stock is pre-incubated with the sera or antibodypreparation (typically for 1 h) that is being evaluated. The sera orantibody preparations are tested undiluted and at incrementally greaterdilutions (typically four to five serial 10-fold dilutions). Infectionof target cells with each virus stock is also performed in the absenceof antibody (no antibody). Virus neutralization is assessed by comparingthe amount of luciferase activity produced in target cells, both in thepresence and absence of antibody. In this embodiment, the results of theassay are interpreted by comparing the ability of each antibody topreferentially block infection of target cells (reduce or eliminateluciferase activity). Virus neutralization activity is quantified bynoting the highest antibody dilution (most dilute) that is able to blocktarget cell infection (e.g., the highest dilution that is able to reducethe luciferase activity produced in the absence of antibody by 50%).

Characterization of Patient HIV-1 (Patient Virus Vs. Standard AntibodyPanel)

In this embodiment, the assay is performed using a target cell line thatexpresses the HIV-1 receptor CD4 plus the HIV-1 co-receptors CCR5 andCXCR4 (HT4/CCR5/CXCR4). Such a cell line is capable of evaluating theneutralizing activity of antibodies for both R5 and X4 tropic viruses.In a related embodiment, the assay is performed using two target celllines. One cell line expresses CD4 plus CCR5 (U-87/CD4/CCR5) and is usedto test R5 tropic viruses. Another cell line expresses CD4 plus CXCR4(U-87/CD4/CXCR4) and is used to evaluate X4 tropic viruses. The virusentry assay is performed by infecting individual target cell cultureswith recombinant virus stocks derived from packaging host cellstransfected with pHIVenv and pHIVluc or pHIVlucΔU3 vectors. In thisembodiment, pHIVenv vectors contain patient virus derived envelopesequences and express HIV-1 envelope proteins (gp120SU, gp41TM). In thisembodiment, viruses from different patient populations (see abovedescription of patient population), and/or different virus populations(see above description for virus population) are used to constructpHIVenv vectors. Pseudotyped HIV derived from pHIVenv vectors areevaluated in the virus entry assay to determine if they are susceptibleto neutralization by a panel of specific, well-characterized antibodypreparations. Such antibodies represent a “standard antibody panel.”Some, but not all, reasonable examples of antibodies that may constitutea standard panel are listed in Table 4. In this embodiment virusneutralization is evaluated using target cells cultured in 96 wellplates. Typically, target cells are plated at 5,000 cells per well forHT4/CCR5/CXCR4 or 10,000 cells per well for U-87/CD4/CCR5 andU-87/CD4/CXCR4 one day prior to infection. Prior to infection, eachpatient derived virus stock is incubated with the each of the antibodypreparations (typically for 1 h) in the standard antibody panel. Thesera or antibody preparations are tested undiluted and at variousdilutions (typically four to five serial 10-fold dilutions). Infectionof target cells with each virus stock is also performed in the absenceof drug (no drug). Virus neutralization is assessed by comparing theamount of luciferase activity produced in target cells, both in thepresence and absence of antibody. In this embodiment, the results of theassay are interpreted by comparing the ability of each antibody topreferentially block infection of target cells (reduce or eliminateluciferase activity). Virus neutralization activity is quantified bynoting the highest antibody dilution (most dilute) that is able to blocktarget cell infection (e.g., the highest dilution that is able to reducethe luciferase activity produced in the absence of antibody by 50%).

Characterization of Patient HIV-1 (Patient Virus V. Patient Antibody)

This example provides a method for detecting within a patient theevolution of a neutralizing antibody response and of viral strains thatevade the neutralizing response. In this embodiment, the assay isperformed using a target cell line that expresses the HIV-1 receptor CD4plus the HIV-1 co-receptors CCR5 and CXCR4 (U87/CD4/CCR5/CXCR4 orHT4/CCR5/CXCR4). Such a cell line is capable of evaluating theneutralizing activity of antibodies for both R5 and X4 tropic viruses.In a related embodiment, the assay is performed using two target celllines. One cell line expresses CD4 plus CCR5 (U-87/CD4/CCR5) and is usedto test R5 tropic viruses. Another cell line expresses CD4 plus CXCR4(U-87/CD4/CXCR4) and is used to evaluate X4 tropic viruses. The virusentry assay is performed by infecting individual target cell cultureswith recombinant virus stocks derived from packaging host cellstransfected with pHIVenv and pHIVluc or pHIVlucΔU3 vectors. In thisembodiment, pHIVenv vectors contain patient virus derived envelopesequences and express HIV-1 envelope proteins (gp120SU, gp41TM). In thisembodiment, different virus populations are used to construct pHIVenvvectors. The different virus populations are derived from serial (i.e.,longitudinal) plasma specimens (i.e., viruses collected from the samepatient at different time points). Pseudotyped HIV derived from pHIVenvvectors are evaluated in the virus entry assay to determine if they aresusceptible to neutralization by a panel of antibodies that are derivedfrom serial plasma specimens from the patient. Thus, in this embodiment,the same patient is the source of both the virus populations and theantibodies. In this embodiment, viruses are evaluated using target cellscultured in, for example, 96 well plates. Typically, target cells areplated at 10,0000 cells per well for the U-87/CD4/CCR5/CXCR4,U-87/CD4/CCR5 and U-87/CD4/CXCR4 cell lines or at 5,000 cells per wellfor the HT4/CCR5/CXCR4 cell line. Target cells are plated on the day ofinfection. Prior to target cell infection, each virus stock ispre-incubated with the sera or antibody preparation (typically for 1 h)that is being evaluated. The sera or antibody preparations are testedundiluted and at incrementally greater dilutions (typically four to fiveserial 5- or 10-fold dilutions). Infection of target cells with eachvirus stock also is performed in the absence of antibody (no antibody).Virus neutralization is assessed by comparing the amount of luciferaseactivity produced in target cells, both in the presence and absence ofantibody. In this embodiment, the results of the assay are determined bycomparing the ability of antibodies derived from the patient atdifferent time points to preferentially block infection of target cells(reduce or eliminate luciferase activity) of the pseudotyped virusesderived from the patient at different time points. Virus neutralizationactivity is quantified by noting the highest antibody dilution (mostdilute) that is able to block target cell infection (e.g., the highestdilution that is able to reduce the luciferase activity produced in theabsence of antibody by 50%). Thus, this embodiment allows one to assayin a patient the co-evolution over time of immunologically distinct HIVstrains and the neutralizing antibody response.

This method was used on a group of 14 treatment-naive patients withprimary HIV infection. Plasma specimens were drawn from each patient at2-4 month intervals (average follow up was 18 months, with a range of6-39 months). Viruses were incubated with serial 5-fold dilutions ofantibody and used to infect target cells that expressed CD4 plus theCCR5 and CXCR4 co-receptors. It was found that 12 of the 14 patientsgenerated strong neutralizing antibody responses to virus. Data from arepresentative patient are provided in FIG. 10. However, each sequentialvirus consistently and rapidly escaped the concurrent neutralizingantibody response. Peak neutralization titers (average 1:1497 dilution,range 1:339-1:4627) developed several months after a virus emerged andthe response remained elevated for many months, to years, thereafter.Neutralizing antibody titers were generally greater to early virusesthan to later viruses from the same patient. Neutralization responses toa heterologous R5 primary virus (JR-CSF) were weak and delayed.Responses to a X4 lab strain (NL4-3) increased over time, but varied inintensity among patients. The magnitude of neutralizing antibodyresponse to autologous virus did not correlate with mean plasma HIV RNAor duration of HIV infection. Thus, the rate of viral neutralizationescape is remarkable and indicates that neutralizing antibody can exerta previously unappreciated level of selective pressure on viralevolution. These data have important implications for natural historyand vaccine development.

Example 7: Identifying HIV-1 Envelope Amino Acid Sequences that ElicitAlter, or Prevent Neutralizing Antibody Responses

This example provides a means and method for identifying HIV-1 envelopeamino acid sequences that elicit/promote, or alter, or prevent antibodymediated neutralization of HIV-1 infection (also referred to as virusneutralization in this application).

Envelope sequences derived from patient samples, or individual clonesderived from patient samples, or envelope sequences engineered by sitedirected mutagenesis to contain specific mutations, are tested in theentry assay to determine co-receptor tropism as described in Example 6.

In one embodiment, antibody mediated neutralization is evaluated inlongitudinal patient samples (viruses collected from the same patient atdifferent time points) is evaluated. For example, virus neutralizationis evaluated prior to vaccination, during a course of vaccination, andat incremental time points after the course of vaccination is completed.In one embodiment, virus neutralization is evaluated for preventativevaccines. In another embodiment, virus neutralization is evaluated fortherapeutic vaccines.

In another embodiment, virus neutralization is evaluated for samplescollected from a large number of different patients. In a furtherembodiment, virus neutralization is evaluated for samples collected froma large number of patients representing different virus and patientpopulations. Such patient populations may include, but are not limitedto, newly infected patients, chronically infected patients, patientswith advanced disease, patients undergoing antiretroviral therapy orimmuno-therapy, vaccinated and unvaccinated individuals. Such viruspopulations may include, but are not limited to, viruses with distinctgenetic characteristics (clade A, B, C, D, E, F, G), viruses susceptibleto antiretroviral drugs, viruses with reduced susceptibility/resistanceto antiretroviral drugs, primary isolates or isolates adapted for growthin cell culture (often referred to as lab-adapted viruses), syncytiainducing (SI) viruses or non-syncytia inducing (NSI) viruses, macrophage(M) tropic viruses, T-cell (T) tropic viruses and dual tropic (M and T)viruses.

-   Genotypic analysis of patient HIV samples

Envelope sequences representing patient sample pools, or clones derivedfrom patient pools, can be analyzed by any broadly available DNAsequencing methods. In one embodiment of the present invention, patientHIV sample sequences are determined using viral RNA purification, RT/PCRand dideoxynucleotide chain terminator sequencing chemistry andcapillary gel electrophoresis (Applied Biosystems, Foster City, Calif.).Envelope sequences of patient virus pools or clones are compared toreference sequences, other patient samples, or to a sample obtained fromthe same patient prior to initiation of therapy, if available. Thegenotype is examined for sequences that are different from the referenceor pre-treatment sequence and correlated to differences in entryinhibitor susceptibility.

-   Antibody mediated neutralization of genetically characterized    viruses

Envelope amino acid sequences that correlate with virus neutralizationare evaluated by constructing envelope expression vectors (pHIVenv)containing a specific mutation on a defined genetic background (e.g.,NL4-3 for X4 tropism, JRCSF for R5 tropism). Mutations may beincorporated alone and/or in combination with other mutations that arethought to modulate virus neutralization. Envelope mutations areintroduced into pHIVenv vectors using any of the broadly availablemethods for site-directed mutagenesis. In one embodiment of thisinvention the mega-primer PCR method for site-directed mutagenesis isused (Sarkar, G. and Summer, S. S., 1990). A pHIVenv vector containing aspecific envelope mutation or group of mutations are tested using thevirus entry assay described in Example 6. Specific antibody preparations(i.e. well-characterized monoclonal of polyclonal antibodypreparations), serum from HIV infected patients, or serum fromvaccinated individuals can be selected to compare neutralizing activity.Antibody neutralization of the virus containing envelope mutations iscompared to antibody neutralization of a genetically defined virus thatlacks the specific mutations under evaluation. The ability of a specificmutation to confer, alter, or prevent antibody neutralization isconfirmed or disproved by introducing the mutation intowell-characterized reference virus and evaluating the antibody mediatedneutralization of the mutant virus in the virus entry assay as describedin Example 6. Observed changes in virus neutralization are attributed tothe specific mutations introduced into the pHIVenv vector.

In one embodiment of the present invention, genetic determinants ofvirus neutralization are identified by evaluating amino acid sequenceswithin the V3 loop of the gp120 surface envelope protein. The amino acidsequences under evaluation are identified by comparing the amino acidsequences of large numbers of viruses that can, or cannot be neutralizedby various well-characterized antibody preparations, patient sera, orsera from vaccinated individuals. Consistent differences in V3 loopamino acid sequences between viruses that can, or cannot be neutralizedare selected for evaluation. Isogenic viruses based on anwell-characterized parental clone (e.g., NL4-3, HXB2, JRCSP) containingspecific “virus neutralization candidate” mutations in the V3 loop ofthe gp120 envelope protein are constructed by site directed mutagenesisand tested for antibody mediated neutralization as described in Example6. Cells expressing CD4 plus CCR5 (e.g., U-87/CD4/CCR5), CD4 plus CXCR4(U-87/CD4/CXCR4), or CD4 plus CCR5 and CXCR4 (HT4/CCR5/CXCR4) areinfected. Amino acid substitutions that change that elicit, alter, orprevent antibody neutralization are deemed important to virusneutralization.

In a related embodiment of the present invention, genetic determinantsof virus neutralization are identified by evaluating amino acidsequences within the entire gp120 surface envelope protein.

In a related embodiment of the invention, genetic determinants of virusneutralization are identified by evaluating amino acid sequences withinthe gp41 transmembrane envelope protein.

Example 8: Measuring Susceptibility to Virus Entry Inhibitors to GuideTreatment Decisions

This example provides a means and method for using virus entry inhibitorsusceptibility to guide the treatment of HIV-1. This example furtherprovides a means and method for using virus entry inhibitorsusceptibility to guide the treatment of patients that have receivedprevious antiretroviral treatment with a virus entry inhibitor. Thisinvention further provides the means and methods for using virus entryinhibitor susceptibility to guide the treatment of patients that havenot received previous treatment with a virus entry inhibitor.

In one embodiment, the susceptibility of patient's viruses to virusentry inhibitors is used to guide the treatment of patients failingantiretroviral regimens that include one or more virus entry inhibitors.Treatment failure (also referred to as virologic failure) is generallydefined as partially suppressive antiviral treatment resulting indetectable levels of virus, which is typically measured in the patientplasma). Guidance may include, but is not limited to, (a) clarificationof available drug treatment options, (b) selection of more activetreatment regimens, (c) clarification of the etiology of rising viralload in treated patients (i.e. poor adherence, drug resistance), and (d)reduction in the use of inactive and potentially toxic drugs. In thisembodiment, resistance test vectors are derived from a patient virussamples and tested for susceptibility to various virus entry inhibitorsusing the phenotypic virus entry assay. Virus entry inhibitors mayinclude, but are not limited to, fusion inhibitors (e.g., T-20, T-1249),co-receptors antagonists (AMD3100, AMD8664, TAK779, PR0542, andpeperidin-1yl butane compounds) and CD4 antagonists (MAb 5A8).Appropriate treatment decisions are based on the results of the virusentry assay (e.g., see FIG. 4B) and additional relevant laboratory testresults and clinical information.

In another embodiment, the susceptibility of patients viruses to virusentry inhibitors is used to guide the treatment of patients that havenot been previously treated with antiretroviral regimens that includeone or more virus entry inhibitors. Guidance may include, but is notlimited to, (a) clarification of available drug treatment options, (b)selection of more active treatment regimens, (c) clarification of thebaseline susceptibility to virus entry inhibitors, and (d) reduction inthe use of inactive and potentially toxic drugs. Determining baselinesusceptibility of virus entry inhibitors in treatment naive patients isimportant for two reasons. First, the natural susceptibility of virusesto entry inhibitors can vary widely (e.g., see FIG. 4A). Second, theincreased use of virus entry inhibitors will undoubtedly result in thegeneration of drug resistant variants that can be transmitted to newlyinfected individuals. In this embodiment, resistance test vectors arederived from a patient virus samples and tested for susceptibility tovarious virus entry inhibitors using the phenotypic virus entry assay.Virus entry inhibitors may include, but are not limited to, fusioninhibitors (e.g., T-20, T-1249), co-receptors antagonists (AMD3100,AMD8664, TAK779, PR0542, and peperidin-1yl butane compounds) and CD4antagonists (MAb 5A8). Appropriate treatment decisions are based on theresults of the virus entry assay and additional relevant laboratory testresults and clinical information.

Example 9: Measuring HIV-1 Co-Receptor Tropism to Guide TreatmentDecisions

This example provides a means and method for using HIV-1 coreceptor(CCR5, CXCR4) tropism to guide the treatment of HIV-1. This examplefurther provides a means and method for using HIV-1 co-receptor tropismto guide the treatment of patients failing antiretroviral drugtreatment. This invention further provides the means and methods forusing HIV-1 co-receptor tropism to guide the treatment of patients newlyinfected with HIV-1.

This example provides a means and method for using virus HIV-1co-receptor tropism to guide the treatment of HIV-1. This examplefurther provides a means and method for using HIV-I co-receptor tropismto guide the treatment of patients that have received previousantiretroviral treatment with a virus entry inhibitor. This inventionfurther provides the means and methods for using HIV-1 co-receptortropism to guide the treatment of patients that have not receivedprevious treatment with a virus entry inhibitor.

In one embodiment, the co-receptor tropism of a patient's virus is usedto guide the treatment of a patient failing antiretroviral regimens thatinclude one or more co-receptor antagonists. Treatment failure (alsoreferred to as virologic failure) is generally defined as partiallysuppressive antiviral treatment resulting in detectable levels of virus,which is typically measured in the patient plasma). Guidance mayinclude, but is not limited to, (a) clarification of the etiology ofrising viral load in treated patients (i.e., poor adherence, drugresistance, change in co-receptor tropism), (b) clarification ofavailable drug treatment options, (c) selection of more active treatmentregimens, and (d) reduction in the use of inactive and potentially toxicdrugs. Monitoring co-receptor tropism in patients receiving treatmentwith CCR5 antagonists has clinical significance, since drug pressure mayresult in a switch to CXCR4 co-receptor tropism. X4 viruses (CXCR4co-receptor tropism) are associated with a poorer prognosis compared toR5 viruses (CCR5 co-receptor tropism). In this embodiment, resistancetest vectors are derived from a patient virus samples and tested forsusceptibility to various co-receptor antagonists using the phenotypicvirus entry-assay. Co-receptor antagonists may include, but are notlimited to, AMD3100, AMD8664, TAK779, PR0542, and peperidin-1yl butanecompounds. Appropriate treatment decisions are based on the results ofthe virus entry assay (e.g., see FIG. 4B) and additional relevantlaboratory test results and clinical information.

In another embodiment, co-receptor tropism of a patient's virus is usedto guide the treatment of patients that have not been previously treatedwith antiretroviral regimens that include one or more co-receptorantagonists. Guidance may include, but is not limited to, (a)clarification of the baseline co-receptor tropism, (b) clarification ofavailable drug treatment options, (c) selection of more active treatmentregimens, (d) reduction in the use of inactive and potentially toxicdrugs. Determining baseline co-receptor tropism has significant clinicalsignificance. Treatment with the appropriate co-receptor antagonist (R5vs. X4 tropism), or antagonists (dual tropism or mixed tropism) islikely to result in a more potent and durable response. In thisembodiment, resistance test vectors are derived from a patient virussamples and tested for susceptibility to various virus entry inhibitorsusing the phenotypic virus entry assay. Co-receptors antagonists mayinclude, but are not limited to, AMD3100, AMD8664, TAK779, PR0542, andpeperidin-1yl butane compounds. Appropriate treatment decisions arebased on the results of the virus entry assay and additional relevantlaboratory test results and clinical information.

TABLE 1 CELLS Cell Receptor 5.25 CXCR4, CD4, CCR5 (not expressed well)BONZO 5.25 .Luc4.M7 CD4, CCR5, BONZO HOS.CD4.CCR5 CD4, CCR5HOS.CD4.CXCR4 CD4, CXCR4 HOS.CD4 CD4, low level expression of CCR5 andCXCR4 HOS HT4 R5 GFP wt CD4, CXCR4, CCR5 HOS.CD4.CCR5.GFP.M7#6* CD4,CXCR4, CCR5 P4.CCRS CD4, CXCR4, CCR5 U87.CD4 CD4 US7.CD4 R5 CD4, CCR5U87.CD4 X4 CD4, CXCR4 MT2 CD4, CXCR4 MT4 CD4, CXCR4 PM1 CD4, CXCR4, CCR5CEm NKr CCR5 CD4, CXCR4, CCR5

TABLE 2 REPRESENTATIVE VIRUSES AND REAGENTS 89.6, SF2 R5-X4/SI ARRRP^(B)92BR014, 92US076 R5-X4/SI/B ARRRP JR-CSF, 91US005 R5/NSI/B ARRRP 91US054SI/B ARRRP NL43, MN, ELI X4/B ARRRP 92HT599 X4 ARRRP 92UG031 R5/NSI/AARRRP (IN-HOUSE) 92TH014, 92TH026 R5/NSI/B ARRRP (IN-HOUSE) 92BR025,93MW959 R5/SI/C ARRRP (IN-HOUSE) 92UG035 R5/NSI/D ARRRP (IN-HOUSE)92TH022, 92TH023 R5/NSI/E ARRRP (IN-HOUSE) 93BR020 R5-X4/SI/F ARRRP(IN-HOUSE) Mabs 2F5, 1577 gp41 TM ARRRP Mabs IG1b12, 2G12, 48D Gp120 SUARRRP Neutralization sera #2, HIV-IG Polyclonal ARRRP CD4-IG Gp120 SUGenentech CD4-IGG2 Gp120 SU Adarc SCD4 Sigma Progenics T20 (DP178) gp41TM Trimeris Rantes, IP1a/b CCR5 SIGMA/ARRRP SDF1a/b CXCR4 SIGMA/ARRRPAMD 3100 CXCR4 AnorMed Dextran sulfate, Heparin Non-specific Sigma aR5(CCR5 co-receptor), X4 (CXCR4 co-receptor) SI (syncytium inducing), NSI(non-syncytium including), A, B, C, D, E, F (envelope clade designation)bAIDS Research and Reference Reagent Program

TABLE 3 PRIMERS TESTED FOR THE AMPLIFICATION OF HIV ENVELOPE RT PRIMERSRT env_N3 5′-GGA GCA TTT ACA AGC AGC AAC ACA GC-3′ RT env 97205′-TTC CAG TCA VAC CTC AGG TAC-3′ RT env 97405′-AGA CCA ATG ACT TAY AAG G-3′ 5′ PCR PRIMERS 5′ env5′-GGG CTC GAG ACC GGT CAG TGG CAA TGA GAG TGA AG-3′ 5′ env_Xho/Pin5′-GGG CTC GAG ACC GGT GAG CAG AAG ACA GTG GCA ATG A-3′ 5′ env_START5′-GGG CTC GAG ACC GGT GAG CAG AAG ACA GTG GCA ATG-3′ 3′ PCR PRIMERS 3′env 5′-GGG TCT AGA ACG CGT TGC CAC CCA TCT TAT AGC AA-3′ 3′ env_Xba/MLu5′-GGG TCT AGA ACG CGT CCA CTT GCC ACC CAT BTT ATA GC-3′ 3′ env_STOP5′-GGG TCT AGA ACG CGT CCA CTT GCC ACC CAT BTT A-3′ 3′ delta CT5′-GAT GGT CTA AGA CGC TGT TCA ATA TCC CTG CCT AAC TC-3′

TABLE 4 ANTI-HIV DRUGS (Panel 1) Drug/Compound Generic name TrademarkManufacturer RT Inhibitors (NRTI, nucleotide analogs) AZT, ZDVZidovudine RETROVIR ® Glaxo/Wellcome 3TC ® Lamivudine EPIVIR ®Glaxo/Wellcome AZT + 3TC ® COMBIVIR ® Glaxo/Wellcome d4T StavudineZERIT ® Bristol-Myers/Squibb ddl Didanosine VIDEX ® Bristol-Myers/SquibbddC Zalcitabine HIVID ® Hoffman LaRoche 1592U89 Ahacavir ZIAGEN ®Glaxo/Wellcome AZT + 3TC ® + 1592U89 TRIZIVIR ® Glaxo/Wellcome (−)FTC(5-fluoro-3TC ®, Coviracil) Emtricitabine Triangle Pharmaceuticals(−)FTC + (+)FTC (50:50) Racimir QuadPharma DAPD (DXG active) AmdoxovirTriangle Pharmaceuticals F-ddA (2-Fluoro-ddA) Lodenosine MedImmuneOncology (US Bioscience) BCH-10652, dOTC (2-deoxy-3-oxa-4- BioChemPharma, Inc. thiocytidine) D-dfFC Triangle Pharmaceuticals (Schinazi) RTInhibitors (NTRTI, nucleotide analogs) bis-POC PMPA (GS-4331) TenofovirGilead Sciences bis-POM PMEA (GS-840) Adefovir dipivoxil Gilead SciencesRT Inhibitors (NNRTI, non-nucleosides) BI-RG-587 Nevirapine VIRAMUNE ®Boehringer&Ingleheim (Roxanne) BHAP PNU-90152T Delavirdine RESCRIPTOR ®Pharmacia & Upjohn DMP 266 (L-743, 726) Efavirenx SUSTIVA ® DupontPharmaceuticals (Avid) MKC442 (Coactinon) Emivirine Triangle/MitsubishiKasei AG-1549 (S1153) (on hold) Capravirine Agouron Pharmaceuticals AZT,ZDV Zidovudine RETROVIR ® Glaxo/Wellcome 3TC ® Lamivudine EPIVIR ®Glaxo/Wellcome AZT + 3TC ® COMBIVIR ® Glaxo/Wellcome d4T StavudineZERIT ® Bristol-Myers/Squibb ddl Didanosine VIDEX ® Bristol-Myers/SquibbddC Zalcitabine HIVID ® Hoffman LaRoche 1592U89 Abacavir ZIAGEN ®Glaxo/Wellcome AZT + 3TC ® + 1592U89 TRIZIVIR ® Glaxo/Wellcome (−)FTC(5-fluoro-3TC ®; Coviracil) Emtricitabine Triangle Pharmaceuticals(−)FTC + (+)FTC (50:50) Racimir QuadPharma DAPD (DXG active) AmdoxovirTriangle Pharmaceuticals F-ddA (2-Fluoro-ddA) Lodenosine MedImmuneOncology (US Bioscience) BCH-10652, dOTC (2-deoxy-3-oxa-4- BioChemPharma, Inc. thiocytidine) D-d4FC Triangle Pharm. (Schinazi) RTInhibitors (NTRTI, nucleotide analogs) bis-POC PMPA (GS-4331) TenofovirGilead Sciences bis-POM PMEA (GS-840) Adefovir dipivoxil Gilead SciencesRT Inhibitors (NNRTI, non-nucleosides) BI-RG-587 Nevirapine VIRAMUNE ®Boehringer/Ingleheim (Roxanne) BHAP PNU-90152T Delavirdine RESCRIPTOR ®Pharmacia & Upjohn DMP 266 (L-743, 726) Efavirenz SUSTIVA ® DupontPharmaceuticals (Avid) MKC442 (Coactinon) Emivirine Triangle/MitsubishiKasei AG-1549 (S1153) (on hold) Capravirine Agouron PharmaceuticalsPNU-142721 Pharmacia & Upjohn DPC-961, -963, -083, -08? DuPontPharmaceuticals SJ-3366 Also entry Samjin Pharmaceuticals inhibitor?BHAP PNU-87201 Atevirdine Upjohn GW420867X (quinoxaline) (2^(nd) gen.HBY 097) Glaxo/Wellcome (Hoechst Bayer) TMC 120 (R147681) Tibotec TMC125 (R165335) Tibotec R86183 tivirapine Janssen PharmaceuticalsCalanolide A SarawakMedichem Protease Inhibitors (PRI) Ro 31-8959Saquinavir-(hgc) INVIRASE ® Hoffman LaRoche Saquinavir-(sgc) FortovaseMK-639 (L-735, 524) Indinavir CRIXIVAN ® Merck Research LaboratoriesABT-538 (A-84538) Ritonavir NORVIR ® Abbott Laboratories AG1343Nelfinavir VIRACEPT ® Agouron Pharmaceuticals 141W94 (VX-478) AmprenavirAGENERASE ® Glaxo-Wellcome/Vertex ABT-378/r Lopinavir/ritonavirKALETRA ® Abbott Laboratories BMS 232, 632 (aza-peptide)Bristol-Myers-Squibb PNU-140690 Tipranavir Pharmacia & Upjohn DMP 450(cyclic urea) Mozenavir Triangle/Avid (ph I/II) TMC 126 (Erickson'scompound) Tibotec G/W433908 (VX-175) amprenavir pro-drugGlaxo/Wellcome/Vertex L756,423 (on hold) Merck PD-178390 (dihydropyrone)Parke Davis (BoehringerIngleheim) ? new candidate Roche DPC 681 and 684DuPont Pharmaceuticals AG-1776 (JE-2147 = KNI-764) AgouronPharmaceuticals Envelope/Receptor Inhibitors T-20 (gp41) PentafusideTrimeris Pharmaceuticals T1249 (gp41) Trimeris Pharmaceuticals D-peptideinhibitor (gp41) small mol. SCH-C Schering-Plough AMD-311 (CXCR4)(bycyclam) AnorMED AMD-3664 (CXCR4) (macrocyclam) AnorMED ALX40-4C(CXCR4) U. PA FP21399 Fuji Pharmaceuticals PRO 542 (gpl20) CD41gG2Progenics Pharmaceuticals PRO-140 (CCR5) MAb CCR5 ProgenicsPharmaceuticals T-22 (CXCR4) (peptide, 18-mer) Met-SDF-1 (CXCR4) TAK 779(CCR5 antagonist) Takeda AOP-Rantes (CCR5) Gryphon Sciences Rantes 9-68(CCR5) CCR5 antagonists 4-(piperidine-1-yl) Merck butane classα-Immunokine-NNS03 (CCR5, α-cobratoxin PhyloMed Corp. CXCR4) IntegraseInhibitors AR-177 Zintevir Aronex Pharmaceuticals Diketo acids MerckResearch Laboratories Nucleocapsid Inhibitors RB 2121 cyclic peptide p7(see PNAS 96: 4886-4891 mimic (1999)) Cl-1012 Achelion PharmaceuticalsRNase H Inhibitor SP1093V (BBNH Fe + 3 derivative) (Parniak) (Panel 2)Generic Name (abbreviation) Brand Name Firm FDA Approval Datezidovudine, AZT RETROVIR ® Glaxo Wellcome March 1987 didanosine, ddlVIDEX ® Bristol Myers-Squibb October 1991 zalcitabine, ddC HIVID ®Hoffman-La Roche June 1992 stavudine, d4T ZERIT ® Bristol Myers-SquibbJune 1994 lamivudine, 3TC ® EPIVIR ® Glaxo Wellcome November 1995saquinavir, SQV, hgc INVIRASE ® Hoffman-La Roche December 1995saquinavir, SQV, sgc Fortovase Hoffman-La Roche November 1997 ritonavir,RTV NORVIR ® Abbott Laboratories March 1996 indinavir, IDV CRIXIVAN ®Merck & Co., Inc. March 1996 nevirapine, NVP VIRAMUNE ® BoehringerIngelheim June 1996 nelfinavir, NFV VIRACEPT ® Agouron PharmaceuticalsMarch 1997 delavirdine, DLV RESCRIPTOR ® Pharmacia & Upjohn April 1997ZDV + 3TC ® COMBIVIR ® Glaxo Wellcome September 1997 efavirenz, EFVSUSTIVA ® DuPont Pharmaceuticals September 1998 abacavir, ABC ZIAGEN ®Glaxo Wellcome February 1999 amprenavir AGENERASE ® Glaxo Wellcome April1999 lopinavir/ritonavir KALETRA ® Abbott September 2000 ZDV + 3TC ® +ABC TRIZIVIR ® Glaxo SmithKline November 2000

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What is claimed is: 1-16. (canceled)
 17. A method for determiningwhether one or more viral species from a population of HumanImmunodeficiency Virus-1 (HIV-1) present in a sample from anHIV-1-infected patient is susceptible to an entry inhibitor comprising:(a) simultaneously, and in a single reaction vessel, amplifying DNAmolecules comprising a portion of the HIV-1 envelope gene of thepopulation of HIV-1 present in the HIV-1-infected patient sample toobtain a plurality of DNA molecules, each encoding the portion of theHIV-1 envelope protein from the population of HIV-1 present in theHIV-1-infected patient sample; (b) simultaneously, and in a singlereaction vessel, inserting the plurality of DNA molecules from step (a)into a first expression vector engineered to express an HIV-1 envelopeprotein in cells resulting in production of a plurality of distinctexpression vectors, each member of which comprises only one of theplurality of DNA molecules, wherein the plurality of distinct expressionvectors express a plurality of variants of the portion of the HIV-1envelope protein that is representative of the HIV-1 species found inthe HIV-1-infected patient sample; (c) transfecting into a plurality offirst cells (i) the plurality of expression vectors from step (b) and(ii) a viral expression vector which lacks the nucleic acid encoding theportion of the HIV-1 envelope protein from step (a) but includes nucleicacid encoding other proteins required for replication of the HIV-1, suchthat the plurality of first cells produce viral particles comprising theHIV-1 envelope proteins having sequences representative of the HIV-1species present in the HIV-1-infected patient sample; (d) contacting afirst portion of the viral particles produced in step (c) with an entryinhibitor and a first plurality of second cells, wherein the secondcells each expresses a cell surface receptor and at least oneco-receptor to which the HIV-1 binds for entry, and wherein either thesecond cells or the viral particles comprise an indicator nucleic acidmolecule which produces a detectable signal following infection of thesecond cells by the viral particles of step (c); (e) contacting a secondportion of the viral particles of step (c) with a second plurality ofthe second cells in the absence of the entry inhibitor; (f) measuringthe amount of the detectable signal produced by the first and secondplurality of second cells from step (d) and step (e) in order todetermine the infectivity of the viral particles in the presence and theabsence of the entry inhibitor, respectively; and (g) comparing theamount of signal measured in step (f), wherein a reduced amount ofsignal measured for the viral particles of step (d) as compared to thesignal measured for the viral particles of step (e) indicates that atleast one species from the population of HIV-1 from the HIV-1-infectedpatient sample is susceptible to the entry inhibitor.
 18. The method ofclaim 17, wherein the portion of the HIV-1 viral envelope polypeptidefrom the patient sample includes at least a portion of the intracellularcytoplasmic tail (CT) domain.
 19. The method of claim 17, wherein theportion of the HIV-1 viral envelope polypeptide from the patient sampledoes not include the intracellular cytoplasmic tail (CT) domain or aportion thereof.
 20. The method of claim 19, wherein the firstexpression vector includes a cytoplasmic tail domain from a geneticallycharacterized HIV-1 virus.
 21. The method of claim 17, wherein step (d)is performed by contacting the viral particles with the entry inhibitorprior to contact with the first plurality of second cells.
 22. Themethod of claim 17, wherein the inhibitor binds or interferes with thebinding of the HIV-1 envelope protein.
 23. The method of claim 17,wherein the inhibitor is an antibody, small molecule, or peptide. 24.The method of claim 23, wherein the antibody binds to CD4, CXCR4, orCCR5.
 25. The method of claim 23, wherein the antibody binds to theHIV-1 envelope protein.
 26. The method of claim 25, wherein the antibodybinds to the CD4 binding site of the HIV-1 envelope protein.
 27. Themethod of claim 25, wherein the antibody binds to the V1/V2 region orthe V3 region of the HIV-1 envelope protein.
 28. The method of claim 25,wherein the antibody recognizes a specific glycosylation pattern of theHIV-1 envelope protein.
 29. The method of claim 25, wherein the antibodybinds to the MPER site of the HIV-1 envelope protein.
 30. The method ofclaim 25, wherein the antibody binds to the CXCR4 binding site or theCCR5 binding site of the HIV-1 envelope protein.
 31. The method of claim17, wherein the indicator nucleic acid molecule comprises a luciferasegene.
 32. The method of claim 17, wherein the receptor comprises atleast one of CD4, and the co-receptor comprises at least one of CXCR4 orCCR5.
 33. The method of claim 17, wherein the plurality of DNA moleculesencoding the portion of the HIV-1 envelope protein encode an HIV-1 gp160envelope polyprotein or an HIV-1 gp120 viral surface envelope proteinand at least a portion of an HIV-1 gp41 transmembrane envelope protein.34. The method of claim 17, wherein the viral expression vectorcomprises an HIV-1 gag-pol gene.
 35. The method of claim 17, wherein thefirst and/or second cells are human cells.
 36. The method of claim 35,wherein the human cells are human embryonic kidney cells, T cells,peripheral blood mononuclear cells, astroglioma cells, or osteosarcomacells.
 37. The method of claim 36, wherein the human embryonic kidneycells are 293 cells, the astroglioma cells are U87 cells, and theosteosarcoma cells are HT4 cells.